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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
GFR
1, 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.
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