(A and D) Whole-mount neurofilament staining of E11.5 Megf8+/+ and Megf8−/− littermates showing the DRG spinal nerves, which are undergrown in the Megf8−/− (arrow). (B and E) Whole-mount peripherin staining of forelimbs from E13.5 Megf8+/+ and Megf8−/− littermates. The radial and ulnar nerves are undergrown in the Megf8−/−embryo. Limbs are outlined with dotted lines. Scale bar (B and E) represents 500 μm. (C and F) Whole-mount neurofilament staining of E11.5 Megf8+/+ and Megf8−/− littermates. The vagus/glossopharyngeal nerves are defasciculated in the Megf8−/− (arrow). (G) Whole-mount neurofilament staining of E11.5 Megf8Flox/Flox and Wnt1-Cre; Megf8Flox/Flox littermates. (H) Quantification of ophthalmic branch phenotype for Megf8−/− (KO), Megf8L1775P/L1775P (L1775P), and Wnt1-Cre; Megf8Flox/Flox (cKO) compared to Megf8+/+ (WT). Left: the number of branches at the nasociliary branch point was significantly greater for KO, L1775P, and cKO embryos. Right: the ophthalmic branch was undergrown in KO, L1775P, and cKO embryos. Four to seven embryos were analyzed per genotype. Error bars represent mean ± s.e.m. *p<0.05, one-way ANOVA. The relative outgrowth was also measured for the maxillary and mandibular branches of the TG for Megf8−/− embryos (not shown). The maxillary branch was slightly undergrown compared to Megf8+/+ (relative outgrowth 0.9, p<0.05) while the mandibular branch was unaffected (relative outgrowth 0.99, p=0.7). To assess defasciculation in the maxillary branch, the relative maxillary area was calculated and no difference was observed between Megf8+/+ and Megf8−/− (relative area 0.94, p=0.2).