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First published online 19 November 2008
doi: 10.1242/dev.026716

Development 136, 85-93 (2009)
Published by The Company of Biologists 2009
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Regulation of enteric neuron migration by the gaseous messenger molecules CO and NO

Sabine Knipp and Gerd Bicker*

University of Veterinary Medicine Hannover, Division of Cell Biology, Institute of Physiology, Bischofsholer Damm 15, D-30173 Hannover, Germany.


Figure 1
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  		Fig. 1. Cellular organization of embryonic enteric nervous system and proposed signal transduction cascades regulating neuronal motility. (A) Schematic drawing of locust embryonic foregut and anterior midgut at 65% E in dorsal view. Midgut is shaded light gray, enteric ganglia and neurons are dark gray. Two of the four migratory pathways are visible on the dorsal midgut. Already developing foregut plexus is omitted for sake of clarity. Double-headed arrow indicates measured distance of enteric neuron migration after 24 hours in vivo culture incubation. ca, cecum; en, enteric neuron; env, esophageal nerve; fc, frontal connective; fg, frontal ganglion; hg, hypocerebral ganglion; ig, ingluvial ganglion; rnv, recurrent nerve. Anterior is towards the left as in following figures. (B) Schematic diagram of CO and NO/cGMP signaling transduction influencing enteric neuron migration. Ca2+-calmodulin (CaM)-activated NOS catalyses the conversion of L-arginine into L-citrulline, thereby releasing NO. NOS activity can be stimulated by applying an excess of arginine or blocked by the inhibitor 7-NI. The diffusible NO binds to the heme moiety in soluble guanylyl cyclase (sGC), thus stimulating synthesis of cGMP. Intercellular diffusing NO can be trapped by the extracellularly acting scavenger hemoglobin. The stimulation of sGC with YC-1 artificially amplifies cGMP production. Heme oxygenase enzymes (HO), such as the HO-2-immunoreactive constitutive isoform, release CO as a by-product during heme degradation. The enzyme activity can be manipulated by its substrate analog hemin or metalloporphyrin inhibitors such as ZnBG and ZnPP-IX. CORM-II is an exogenous CO donor. CO competes with NO for binding to sGC (blunt tip), leading only to a rather modest increase in the cGMP level.

 

Figure 2
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  		Fig. 2. Immunochemical investigation of cGMP formation and the expression of NOS in the embryonic enteric nervous system. (A,B) Confocal fluorescence images of migrating enteric neurons on the midgut at 65% E in a so-called `tissue blot' preparation. Neurons were marked immunocytochemically for anti-cGMP (red) and anti-acetylated {alpha}-tubulin (green). (C) Merged image of A and B. m, gut musculature. Scale bar: 50 µm. (D) Immunoblot analysis with an antibody against universal NOS in homogenates of different parts of embryonic gut and in the CNS (65-70% E). The antibody recognizes a protein of ~135-140 kDa and a second, somewhat lighter protein band in the fore- and midgut cytosolic fraction. Bottom lanes provide an acetylated {alpha}-tubulin band as loading control. For quantification, the ratio of the NOS signal to the {alpha}-tubulin signal was calculated and averaged for all probed blots (mean NOS ratios, n=4, s.e.m. of the ratios are: ±0.07 MG, ±0.04 MG-M, ±0.01 FG, ±0.08 CNS). The NOS ratio directly below the lanes corresponds to the actual signals of the shown western blot. MG, midgut; MG-M, membranous part of midgut (both midgut homogenates included adjacent hindgut); FG, foregut; CNS, central nervous system, including ventral nerve cord.

 

Figure 3
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  		Fig. 3. Quantitative evaluation of enteric neuron migration after chemical manipulation of the NO/cGMP pathway and HO enzymes. Bar plots show average migration distance covered by the leading enteric neurons during 24 hours of in vivo culture. Data result from at least two independent experiments, each normalized to the mean of the corresponding control. (A) Inhibition of NOS with 500µM 7-NI or scavenging NO with 500µM hemoglobin result in a significant reduction of migration on dorsal migratory pathways. (B) Excess of NOS substrate L-arginine (2 mM) or stimulation of sGC with 65µM YC-1 revealed no difference of migration compared with the control. (C) Inhibition of CO releasing HO enzymes with 10µM ZnPP-IX or 5µM ZnBG lead to a significant acceleration of enteric neuron migration. Activating HO with 100 µM hemin or applying CORM-II (20µM) resulted in a significant reduction of average migrated distance. Application of inactivated CORM-II (iCORM-II, 20µM) did not affect migration. (D) Enteric neuron viability after 24 hours of in vivo culture with 100µM hemin or 200µM CORM-II. 100% represents total number of counted enteric neurons. Error bars are ±s.e.m. The numbers of experimental gut preparations are indicated in the bars. A Wilcoxon Mann-Whitney test was employed for statistical comparisons. ***P<0.001; **P<0.005; *P<0.05.

 

Figure 4
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  		Fig. 4. Enteric neuron migration under influence of neurochemicals affecting heme oxygenase catalyzed CO production. Immunofluorescence images of enteric neuron migration on individual guts after 24 hours in vivo culture. Dissected guts were stained with cGMP antiserum. Composed images are resulting from microphotographs of several focal planes, that were converted to gray scale, inverted for fluorescence intensity and arranged in Adobe Photoshop. (A) Control conditions. (B) Inhibition of HO enzymes by 5 µM ZnBG. (C) Activation of HO with excess of its substrate analogue hemin (100 µM). (D) Exogenous carbon monoxide (CORM-II, 20 µM). Broken lines indicate foregut-midgut boundary. Arrows indicate furthest migrated enteric neuron. ig, ingluvial ganglion. Scale bar: 500 µm.

 

Figure 5
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  		Fig. 5. Western blot analysis of HO in the enteric nervous system. (A) Immunoblots obtained from whole guts at 65% E with enteric nervous system attached (ENS) and from dissected CNS. HO-2 antibody recognizes proteins of ~30-35 kDa. In CNS and cytosolic fractions an additional, slightly heavier protein band is enriched in the membrane fraction of homogenates. Both membrane (left) and cytosolic (right) fractions derive from the same preparation. Bottom lanes provide an acetylated {alpha}-tubulin band as loading control. For quantification, the ratio of the HO-2 signal to the {alpha}-tubulin signal was calculated for each lane (n=3, s.e.m. of the ratios are: ±0.29 membrane ENS, ±0.17 membrane CNS, ±0.08 cytosol ENS, ±0.03 cytosol CNS). (B) Immunoblotting of recombinant rat HO-2 protein and membrane fraction of ENS at 65% E. The blots were probed either with the antibody against HO-2 (left, anti-HO-2) or corresponding antibody solution pre-adsorbed with HO-2 protein (right, anti-HO-2, pre-adsorbed). Arrowheads in A and B indicate distinct HO bands.

 

Figure 6
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  		Fig. 6. Immunocytochemical detection of HO in the enteric nervous system. (A,B) Immunocytochemical labeling of the enteric neurons migrating on the midgut at 65% E, using the antiserum in a whole-mount (A) and tissue blot (B) preparation. 1% BSA was added to the blocking solution in B. (C) HO-2 immunocytochemistry at 95% E on a whole-mount midgut. Enteric neurons have left the four migratory pathways and established the midgut plexus. At this stage, other tissues such as gut musculature show similar labeling intensity. (D) ENS at the same developmental stage as in C but stained for acetylated {alpha}-tubulin. A, C and D are composed images from microphotographs of several focal planes. m, gut musculature. Scale bars: 50 µm.

 

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