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Fig. 4. Comparison of the expression patterns of Xotch, chd and
shh. In situ hybridisation of (A,D,G,J) Xotch, (B,E,H,K)
chd and (C,F,I,L) shh. (A-C) Vegetal view of an early
gastrula, stage 10.25, dorsal side up, showing (A) the distribution of
Xotch mRNA in the dorsal marginal zone, (B) the expression of
chd in the organiser and (C) shh transcripts, first seen at
this stage in the organiser and found in a subdomain of the chd
territory. (D-F) Vibratome section in the sagittal plane of an early gastrula
showing (D) the expression of Xotch in the epithelial and
subepithelial layers of the organiser, (E) the expression of chd in
the epithelial and subepithelial layers and in the deep zone of the organiser
and (F) a patch of cells expressing shh in the subepithelial layer of
the organiser, with some faint staining in the epithelial layer. The
arrowheads in D,E,F point to the dorsal blastoporal groove. Distinctions in
the cellular composition of the gastrula organiser followed the criteria of
Hausen and Riebesell (Hausen and
Riebesell, 1991 ). (G) Late gastrula/early neurula embryo, dorsal
view, anterior up, showing the distribution of Xotch transcripts.
Interestingly, we observed asymmetries in several embryos, with higher levels
of Xotch mRNA on the right side. The yellow line indicates the plane
of section shown in J. (H) Dorsal view of a late gastrula, anterior up,
showing the expression of chd in the involuted cells. (I) Dorsal view
of a late gastrula, anterior up, showing the expression of shh in the
dorsal midline. (J) Transverse vibratome section at the level of the posterior
trunk of the same embryo as in G. Xotch transcripts are found in
proneural domains (p) of the neural plate, presomitic mesoderm (s) and in the
developing floor plate (f) but are not found in the notochord (n). Notice
higher levels of Xotch mRNA in the proneural and presomitic domains
on the right side. More anterior sections also showed the presence of
Xotch transcripts in prospective floor plate cells, although at lower
levels, and their absence from the notochord (not shown). (K) Sagittal section
of the same embryo shown in H, anterior to the left; the blastopore is at the
right margin. Chd transcripts are distributed along the notochord.
(L) Sagittal section of the same embryo shown in I, anterior to the left; the
blastopore is at the right margin. Shh transcripts are found in the
three dorsal midline layers, in a dorsal to ventral gradient.
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transcript
was used as internal standard. (B-D'') In situ hybridisation of
shh. (E-F',G,H) In situ hybridisation of plvs.
(F'') Immunofluorescence revealing the Myc-tag epitope. Whole embryos in
B,C,D,E,F,G,H are shown in dorsal views, anterior up. The nuc-lacZ
(nuc-sßgal) tracer is visualised by the turquoise X-gal staining. (B)
Stage 13 embryo injected with 2.5 ng of nuc-lacZ. (C) Stage 13 embryo
injected with 1 ng of notchICD. (D) Stage 13 embryo
injected with 2 ng of X-su(H)DBM shown at high
magnification to appreciate the down-regulation of shh on the
injected side (right). (B') Transverse section of a stage 14 embryo
injected with 2.5 ng of nuc-lacZ. (C',C'') Transverse
section of a stage 14 embryo injected with 1 ng of
notchICD. Arrow points to the expanded floor plate domain
of shh on the injected side, while loss of notochordal tissue is
indicated by an asterisk. (D',D'') Transverse section of a stage 14
embryo injected with 2 ng of X-su(H)DBM. Arrow points to
the depletion of shh transcripts from the floor plate domain on the
injected side. The broken line in C',D' indicates the notochord
contour determined by the Nomarski interference contrast view shown in
C'',D'', respectively. (E) Stage 12 embryo injected with 1.5 ng of
nuc-lacZ. (F) Stage 12 embryo injected with 1 ng of
notchICD. (E') Transverse section of a stage 14
embryo injected with 1.5 ng of nuc-lacZ. (F',F'')
Transverse section of a stage 14 embryo injected with 1 ng of
notchICD. The arrow in F' indicates the expanded
floor plate domain of plvs. The broken lines in F'' outlines the
notochord and neuroectodermal contour of the section shown in F. Ectopic floor
plate cells that inherited the notchICD mRNA, as revealed
by the Myc-tag epitope, express plvs. (G) Stage 13 embryo injected
with 2 ng of nuc-lacZ shown at high magnification. (H) Stage 13
embryo injected with 2 ng of X-su(H)DBM shown at high
magnification to appreciate that plvs expression is strongly reduced
on the superficial layer of the injected side (right) corresponding to the
floor plate domain, while a deeper staining, corresponding to the notochord,
remains.
