Fig. 4. The esophagi of Sulf1^-/-;Sulf2^-/- mice have diminished neuronal innervation and enteric glial cells. The lower-half thoracic segments of the esophagus were immunolabeled either on cross-sections or by whole-mounts with various antibodies to examine neural innervation. (A) Comparable TuJ1 staining and expression of GFR1, p75 and GDNF in Sulf1^+/-;Sulf2^+/- control esophagi and Sulf1^-/-;Sulf2^-/- esophagi at E14.5 (n=3). (B) Reduced neural innervation at the esophageal smooth muscle layer in Sulf1^-/-;Sulf2^-/- esophagi at E18.5 (n=4). The whole esophagus was mounted on the glass slide after whole-mount immunohistochemistry with the TuJ1 antibody. One side of the flattened esophagus was photographed. The number of neurons (cell body marked by ^*) on one side of a 1-mm longitudinal segment was counted and four non-overlapping segments were counted for each esophagus. Numbers presented are the averages of the neural number per 1-mm segment. The innervation density at the smooth muscle layer was calculated by dividing the total number of innervating TuJ1⁺ neurites (white arrowheads) on cross-sections by the circumference of smooth muscle. The circumference of the smooth muscle was not different between control and Sulf1^-/-;Sulf2^-/- esophagi at E18.5 (control, 0.81±0.09 mm; mutant, 0.83±0.07 mm). A minimum of five serial cross-sections (200 µm apart) of each esophagus were quantified. The smooth muscle innervation was also shown by p75 staining (white arrowheads). Sulf1^-/-;Sulf2^-/- esophagi had the same number of neurons as the control, whereas the smooth muscle innervation was reduced in Sulf1^-/-;Sulf2^-/- esophagi. (C) Reduced esophageal innervation and enteric glial cells in Sulf1^-/-;Sulf2^-/- esophagi at P15. Arrowheads indicate the TuJ1⁺ neurites innervating the smooth muscle of muscularis mucosae. Arrows point to the enteric glial cells located between the skeletal muscle layers. The circumference of smooth muscle on cross-sections of Sulf1^-/-;Sulf2^-/- esophagi (1.37±0.18 mm) was 20% longer than that of littermate controls (1.15±0.13 mm). A minimum of 28 sections from four independent controls or Sulf1^-/-;Sulf2^-/- mice were counted. Data shown in the bar graphs represent the number of TuJ1⁺ neurites innervating smooth muscle, innervation density and the average number of enteric glial cells per cross-section. Statistics were calculated by two-tailed Student's t-test. Scale bars: 100 µm.