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doi: 10.1242/10.1242/dev.00433

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GDNF availability determines enteric neuron number by controlling precursor proliferation

¹ Departments of Pediatrics and Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, MO 63110, USA
² Departments of Pathology and Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA
³ Departments of Physiology and Medicine, Medical College of Virginia of Virginia Commonwealth University, Richmond, VA 23298, USA
⁴ Laboratory for Neuronal Differentiation and Regeneration, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 Japan

View larger version (32K): [in a new window]   Fig. 2. Quantitative analysis of adult mouse small bowel and colon ENS structure. (A) Neuron number in wild-type and mutant mouse myenteric and submucosal plexus. (B) Cell size data. (C) Fiber density data for acetylcholinesterase-stained myenteric plexus. Error bars show s.e.m. *Differs significantly from wild type (P<0.05).

View larger version (34K): [in a new window]   Fig. 5. Enteric neuron number is not influenced by Bax or Bid deficiency. Quantitative neuron counts in the myenteric and submucosal plexus of wild-type, Bax–/–, Bid–/–, Gdnf+/–, Gdnf+/–/Bax–/– and Gdnf+/–/Bid–/– mice are presented. Although GDNF heterozygosity caused reduced neuron numbers, Bax and Bid-deficient animals had normal numbers of enteric neurons. In addition, Bax and Bid deficiency did not rescue the reduction in neurons seen in the GDNF heterozygous mice.

View larger version (170K): [in a new window]   Fig. 1. Wholemount small bowel myenteric plexus staining for Gdnf+/–, Gdnf+/–/Nrtn–/–, Nrtn–/– and wild-type mice. (A,B) Cuprolinic Blue staining highlights clustered neuronal cell bodies (red arrows) without showing neuronal processes. Gdnf+/– mice (B) have fewer neurons, but normal neuronal cell size. (C-F) Acetylcholinesterase stains both neuronal cell bodies and fibers. Gdnf+/– mice (D) have a normal density of acetylcholinesterase-stained fibers compared with wild-type (C). Nrtn–/– (E) and Gdnf+/–/Nrtn–/– (F) mice have an obvious loss of small acetylcholinesterase-stained neuronal fibers. Black arrowheads identify small fibers and fiber bundles in Gdnf+/– and Gdnf+/–Nrtn–/– mice (D,F). Scale bar: 100 µm.

View larger version (108K): [in a new window]   Fig. 3. GFR1 and GFR2 expression in the gut. (A,B) E14 gut expresses GFR1 (red arrows, FITC) but not GFR2 (FITC). Yellow arrows (B) show PGP9.5-expressing neural crest cells in the gut (Cy3 secondary), but there is no detectable GFR2 staining. Newborn (C,D) and adult (E,F) small bowels express GFR1 (FITC) and GFR2 (FITC), but GFR2 is much more easily detected in adult (F) than in newborn mouse gut. Scale bar: 100 µm.

View larger version (149K): [in a new window]   Fig. 4. Apoptotic cells are easily detected in the DRG, but not in the gut. (A,B) Activated caspase 3 immunohistochemistry easily identifies apoptotic cells in E12 DRG (A, white arrows), but not in the E12 ENS (B). Auto-fluorescent nucleated red blood cells are also seen (red arrows) in both the gut and DRG. (C,D) Sections of the gut at E14 (C) and P0 (D) were stained with antibodies to activated caspase 3 (Cy3 secondary antibody) and with antibodies to PGP9.5 (FITC secondary antibody) to show ENS precursors. Activated caspase 3 stained cells were not seen within the ENS at any age examined. Scale bars: in A, 100 µm for A; in B, 100 µm for B-D.

View larger version (35K): [in a new window]   Fig. 6. GDNF heterozygous mice have a reduced rate of ENS neuronal precursor proliferation. (A,B) BrdU (FITC) and PGP9.5 (Cy3) double labeling in the E12 gut. The red arrow indicates a PGP9.5-positive cell that had incorporated BrdU. Blue arrows indicate PGP9.5-positive cells that have not incorporated BrdU. (C) Quantitative analysis of the rate of ENS neuronal precursor proliferation. *P=0.01. Scale bar: 20 µm.

View larger version (34K): [in a new window]   Fig. 7. Gdnf+/–, Nrtn–/–, Gdnf+/–/Nrtn–/–, Gfra1+/– and Ret+/– mice all have abnormal intestinal contractility and reduced VIP and substance P release. Contractility of intestinal circular (A,B) and longitudinal (C,D) muscle in response to electric field stimulation. Different colored bars, as indicated in H, represent distinct mouse genotypes. (A-D) Intestinal segments from the small bowel (A,C) or colon (B,D) were stimulated with a 1 minute electric field stimulus at 80 V and 0.5, 1.0, 5.0 or 10.0 Hz. Contractile strength was measured with force transducers. Small bowel circular (A) and longitudinal (C) muscle, as well as colon circular muscle (B), contract during electric field stimulation. Colon longitudinal muscle relaxes during field stimulation, and then has a rebound contraction. The colon longitudinal muscle data (D) plots the rebound contraction phase. The relaxation phase that occurs during electric field stimulation was also reduced in all genotypes compared with wild-type mice, but the data are omitted to simplify the figure. Release of VIP (E,F) and substance P (G,H) from the small intestine (E,G) or colon (F,H) was measured either in the basal state or after electric field stimulation. The bars for transmitter release in response to electric field stimulation represent the increase in transmitter release over baseline. Error bars show s.e.m.. All of the mutant genotypes differ from wild type for all of the contraction and transmitter release parameters measured (P<0.05).