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Fig. 4. Peripheral and enteric nervous system defects in Foxd3^flox/-; Wnt1-Cre embryos. (A-H) β-galactosidase activity detected by X-Gal staining in control and mutant mouse embryos. (A,B) Lateral view of hindlimbs of 14.5 dpc embryos. In control embryos, development of cutaneous nerves is obvious and the developing sciatic nerve is visible (A). None of these structures is seen in Foxd3 mutant embryos (B). (C,D) Dorsal view of the trunk of a control embryo at 14.5 dpc shows the segmented development of spinal nerves radiating out from the developing spinal cord (C). This is not seen in mutant embryos (D). (E,F) Lateral view of a 14.5 dpc embryo shows coils of the developing gastrointestinal tract. In the control embryo, cells of the ENS stain blue on the outside of the gut coils (E). In the Foxd3 mutant embryo, no blue staining of the ENS is visible and the absence of cutaneous peripheral nerves is apparent (F). (G,H) Dissected gastrointestinal tracts from 17.5 dpc embryos. In the control, the entire extent of the gut is ensheathed in NC-derived cells making up the ENS (G). In the mutant, there are no β-gal-positive cells around the outside of the gut (H). Blue staining inside the lumen is background (arrowhead). (I,J) Immunofluorescence for PGP9.5, a protein enriched in neurons, shows the location of the ENS neurons in cross-sections of the intestine in the control embryo (I), whereas no PGP9.5 signal is detected in the mutant (J). PGP9.5 signal is red; sections were counterstained with DAPI (blue) to reveal nuclei. cn, cutaneous nerves; ENS, enteric nervous system; sc, sciatic nerve; sn, spinal nerves; st, stomach.

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