1. Volume 139, Issue 4, Pages 1344-1354 (October 2010)
Loss of Lsc/p115 Protein Leads to Neuronal Hypoplasia in the Esophagus and an Achalasia-Like Phenotype in Mice 
Eugen Zizer, Sven Beilke, Tobias Bäuerle, Kerstin Schilling, Ursula Möhnle, Guido Adler, Klaus–Dieter Fischer, Martin Wagner 
Gastroenterology 
Volume 139, Issue 4, Pages (October 2010)
DOI: /j.gastro
Copyright © 2010 AGA Institute Terms and Conditions

2. Figure 1
Expression of lsc/p115 in the esophagus. (A) Western blot analysis reveals lsc/p115 in the esophagus, stomach, colon, and spleen, but not in the small intestine. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as loading control. (B) RNA in situ hybridization reveals mRNA expression in the epithelial (arrows) and muscle layers (arrowheads); (C) sense probe control. (D) Immunofluorescence shows lsc/p115 (green) surrounding the β-tubulin class III–positive neurons (red). (E) Lsc/p115 expression (red) overlaps with GFAP staining (green). Scale bars, 100 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
Reduced muscle layer in lsc/p115-deficient mice. Macroscopic examination shows marked dilatation of the esophagus above the LES (arrows) in a (A) control and a (B) 1-year-old lsc/p115-deficient mouse. (C) Volumetric computed tomography scan of a 40-week-old lsc/p115-deficient mouse shows the dilatation in the distal part of the esophageal cavity (arrow) in the 3-dimensional surface reconstruction. H&E staining shows the reduced thickness of the muscle layer in (E) lsc/p115-deficient mice compared with the (D) control. (F) Measurement of the muscle layer 10 mm proximal to the LES shows significant decrease both in 20- and 50-week-old lsc/p115-deficient mice (light grey bars) as compared with the control (black bars). Furthermore, the muscle layer is reduced in 50-week-old lsc/p115-deficient mice as compared with 20-week-old lsc/p115-deficient mice. Scale bars, (A and B) 1 cm and (D and E) 100 μm. Quantitative data are presented as the mean and standard deviation. *P < .05 (Wilcoxon rank sum test with continuity correction).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

4. Figure 3
Analyses of muscle cell differentiation in lsc/p115-deficient mice. (A) Masson–Golden trichrome staining of the control depicts several layers of connective tissue stained in green (arrows). (B) These layers are present in lsc/p115-deficient mice; however, they are reduced markedly (arrows). Western blot analyses show comparable expression of (C) fast skeletal myosin and (D) smooth muscle myosin in 3 individual control mice as compared with lsc/p115-deficient mice (lsc −/−). (E) Myogenin shows comparable protein levels in control mice and lsc/p115-deficient mice (lsc −/−) in all sections of the gastrointestinal tract evaluated. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) served as loading control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

5. Figure 4
Esophageal manometry indicates severe motility disorder in lsc/p115-deficient mice. (A) Swallow-induced peristalsis in the wild-type control with similar responses in the 3 simultaneous recordings 21, 13, and 5 mm above the LES. (B) Lsc/p115-deficient mouse responds with uncoordinated, high-pressure contractions in the recordings of the esophageal body. (C and D) LES relaxation in the control and lsc/p115-deficient mouse, respectively. (D) The knockout animal has a markedly increased resting pressure, incomplete relaxation, and prolonged recovery to the baseline in the LES recording. (E) Summary of LES baseline and relaxation pressures in 14-week-old and 1-year-old control mice (dark bars) and lsc/p115-deficient animals (light grey bars). Quantitative data are presented as the mean and standard deviation. *P < .05. (E) At least 4 animals were evaluated for each group.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
Impaired neurite density in lsc/p115-deficient mice. (A–D) Whole-mount staining of the muscle layer with β-tubulin class III indicates reduced neurite density in the thoracic esophagus and the LES of the knockout mice (B and D, respectively) as compared with the control (A and C, respectively). (E) Quantitative evaluation of the number of β-tubulin class III–positive neurites in these whole-mount stainings shows reduced neurite density both in the LES and the thoracic esophagus of lsc/p115-deficient mice (light grey bars) as compared with the control (dark bars). (F) mRNA expression of β-tubulin class III is reduced to approximately 50% of the control level (dark bars) in the knockout mice (light grey bars). (A–D) Scale bars, 100 μm. Quantitative data are presented as the mean and standard deviation. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

7. Figure 6
Reduction of inhibitory and excitatory neurons in lsc/p115-deficient mice. (A) Expression of nNOS is decreased in the LES of 3 lsc/p115-deficient mice (lsc −/−) as compared with the controls upon Western blot analyses. Expression of (B and C) nNOS and (D and E) ChAT was evaluated with immunohistochemical staining of (B and D) control and (C and E) lsc/p115-deficient mice. Inserts depict high-power magnifications. (F) Quantitative evaluation shows a significant decrease of the number of ViP- and ChAT-positive neurons in the knockout mice (light grey bars) as compared with the control (dark grey bars). The difference in the number of nNOS-positive neurons was not significant. (B–G) Scale bars, 150 μm. Quantitative data are presented as the mean and standard deviation. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

8. Figure 7
Reduced glia cells and NGF expression in lsc/p115-deficient mice. GFAP-positive glia cells are reduced in (B) lsc/p115-deficient mice as compared with the (A) control. (C) Quantitative RT-PCR indicates decreased GFAP–mRNA levels in the knockout animals (light grey bars) as compared with the control (dark grey bars). NGF expression is reduced at the (D) mRNA level and upon (E) Western blot analysis. (A and B) Scale bars, 100 μm. Quantitative data are presented as the mean and standard deviation. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions