Figure 4.

Disruption or elimination of the heparin binding site (HBS) domain abolishes FGF8 bioactivity. (A) Amino acid alignment of wild-type (wt) and the patient-specific 15 bp deletion region of FGF8. Red characters indicate that conserved five residues around HBS domain (glycine box). The missing five amino acid residues are indicated by ‘–’. (B) Injection constructs. Two different deletions of the HBS region have been generated by PCR. The first faithfully represents the 15 bp deletion from the proband, while the other represents the full deletion of the HBS as a control. (C) Lateral view of the injection phenotype of FGF8 mRNA at 24 h after fertilization (hpf). Small inner images of each Type are visualized by a ventral view of the forebrain. Decreased size of the eye (arrowhead) and short trunk (arrow) are the major phenotypic findings. Type I with normal development, Type II with only reduced eye size, Type III with reduced eye and short trunk, Type IV with reduced eye and extremely short trunk size. (D) Bioactivity measurements were made by counting embryos based on their phenotypes (Type I–IV) and calculating their percentage. Quantified embryos for this study have been described in Supplementary Material, Table S3.