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.