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Fig. 8. Sulfated GAGs are necessary for transmission of the Nodal signal from
the node to the LPM. (A,J) Schematic representation of
proteoglycans from normal (A) or chlorate-treated (J) mouse embryos. A serine
residue (yellow) of the core protein (orange) is attached to the GAG chain
(blue curved line), which is sulfated (blue circles) under normal conditions
but not in cells treated with chlorate. (B-D,K-M) Transverse
frozen sections of embryos cultured to the six-somite stage in the absence
(B-D) or presence (K-M) of 15 mM sodium chlorate were subjected either to
immunofluorescence analysis with antibodies to HS (B,K) or to CS (C,L) or to
staining with Alcian Blue (D,M). (E-G,N-P) In situ hybridization
for Nodal (E,N), GDF1 (F,O) or Cryptic (G,P) mRNAs
in embryos cultured in the absence (E-G) or presence (N-P) of chlorate.
(H,I,Q,R) Expression vectors for Nodal and GFP
were co-injected into the right LPM of embryos at the headfold stage, which
were then cultured to the six-somite stage in the absence (H,I) or presence
(Q,R) of chlorate. The cultured embryos were examined for GFP fluorescence
(H,Q) and then subjected to in situ hybridization for Nodal mRNA
(I,R). It should be noted that the region of Nodal expression was
much broader than the area expressing GFP (I,R), which is due to a competence
of LPM for Nodal auto-activation. (S) The number and percentage of
embryos with (blue) or without (white) Nodal expression in LPM after
culture in the absence or presence of chlorate. The difference between the two
culture conditions was statistically significant (P<0.001) by the
two-tailed Fisher's exact probability test. Scale bars: 100 µm in B-D,K-M;
200 µm in E-I,N-R.
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