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Fig. 4. Ndst1; Shh interact genetically and biochemically. Ptch1 protein
expression is reduced in the developing face of
Ndst1–/– embryos and Fgf signalling is also
affected. (A-C) Craniofacial morphology in E15.5 embryos. (A)
Ndst+/– and (B) Shh+/–
littermate controls. (C) Some Ndst1; Shh compound heterozygous mice
showed hypoplastic maxillary processes (arrow). (D,E) Haematoxylin/Eosin
staining of horizontal sections of embryos in A,C, respectively. The olfactory
epithelium (ot) is absent in the Ndst1, Shh compound heterozygous
mouse, and development of the eye lens (el) is also affected, both resembling
features found in the Ndst1–/– mutant embryos
(Fig. 2F,H). (F) Wild-type
embryo, (G) Ndst1–/– embryo. (F,G) Reduced
expression of the Shh receptor Ptch1 in the frontonasal process of
Ndst1–/– mutant E15.5 embryos, as assessed by
immunohistochemical staining. Three wt and mutant embryos were analyzed,
representative sections are shown. Green fluorescence indicates Ptch1
expression. (H) More AP-Shh binds to E14.5 wild-type GAGs (red line) than to
GAGs isolated from mutant embryos (blue line). Equal amounts of GAGs isolated
from these embryos were coupled with Affi-Gel, loaded with recombinant,
soluble alkaline phosphatase fused to Shh (AP-Shh) followed by elution with
increasing NaCl concentrations ranging from 0-1 M in 50 mM increments. (I,J)
Impaired Fgf signalling in Ndst1 mutant embryos. (I) Twenty-five
percent of all Ndst1–/– embryos showed
developmental defects of the first branchial arch, including agnathia strongly
reminiscent of neural- and neural-crest specific Fgf8 mutant mice
(Trumpp et al., 1999). One
representative E18.5 Ndst1 mutant embryo is shown. (J) Fgf-dependent
Erk1/2 phosphorylation is significantly reduced in Ndst1 mutant
mesenchymal cells isolated from two mutant embryos (top and middle). Cultured
cells derived from facial mesenchyme of two E14.5 wild-type and two mutant
littermates were stimulated with 10% serum in DMEM or 10 ng/ml Fgf2 in DMEM
for 5 minutes before analysis. Phosphorylation of Erk1 and Erk2 (p-Erk1 and
p-Erk2) were observed in the wild type after addition of serum or Fgf2;
however, Fgf2 addition alone failed to stimulate Erk1/2 phosphorylation in the
Ndst1 mutant cells. Control refers to no stimulation. (Bottom)
Detection of total Erk1 and Erk2 with anti-Erk antibodies.
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