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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00612

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Nitric oxide and cyclic nucleotides are regulators of neuronal migration in an insect embryo

School of Veterinary Medicine Hannover, Cell Biology, Bischofsholer Damm 15, D-30173 Hannover, Germany

View larger version (27K): [in a new window]   Fig. 1. NO induced cGMP-immunoreactivity of MG neurons during midgut plexus development. Guts were incubated with SNP and then immunostained with an anti-cGMP antiserum. (A) Images were drawn from individual preparations. Each panel shows a dorsal view of the embryonic gut: ingluvial ganglion (ig), the midgut is marked in gray; mg indicates midgut neurons. For the sake of clarity, the caeca are not shown. Scale bar: 200 µm. (B) Appearance of NO-induced cGMP-IR in MG neurons. The total number of MG neurons was calculated from anti-acetylated -tubulin staining which labels all MG neurons. The percentages of cGMP-positive MG neurons during different embryonic stages are shown. The mean values and s.e.m. were calculated for each developmental stage (n=5). At 55% E, almost no cGMP-IR was found in the premigratory population of MG neurons. When migration started at about 60% E, all migrating MG neurons showed strong levels of anti-cGMP staining. The MG neurons exhibited cGMP-IR throughout the phase of migration (60-70% E) and continued to show high levels of anti-cGMP staining in the phase of lateral axon branching and the formation of terminal processes on the midgut musculature (70-85% E). When the midgut plexus acquired a mature innervation pattern (90 and 95% E), there was a rapid decrease of cGMP-IR.

View larger version (50K): [in a new window]   Fig. 2. Developmental expression of NO induced cGMP-IR in MG neurons and NADPH diaphorase staining of the midgut epithelium. (A) At about 60% of embryonic development (% E), the MG neurons (mg) formed a cellular packet at the foregut-midgut boundary (vertical line indicates boundary). Caeca (ca) were not stained. At this stage, all MG neurons began to show strong anti-cGMP staining. In the ingluvial ganglion (ig), some neurons expressed sGC activity (lateral view). (B) Between 60 and 65% E, the first MG neurons started to migrate posteriorly on the midgut surface. All MG neurons showed high levels of anti-cGMP staining. (C,D) Between 60 and 65% E, NO-sensitive sGC activity was expressed in the cell body and the advancing processes of the leading MG neurons. (E) At 60% E, first NADPH-diaphorase staining was present in distinct cells of the midgut. The inset of the designated area shows an example of NADPH-diaphorase-positive cellular staining. The first appearance of the diaphorase staining was coincident with the onset of MG neuron migration (compare to A with B). (F) Anti-cGMP-IR at 65% E; lateral view. At this stage, anti-cGMP IR was present in cells of the ingluvial ganglion, the enteric nerves and the foregut neurons (fg). Some of the midgut neurons migrated laterally to form a nerve ring near the foregut-midgut boundary. (G) At 70% E, the MG neurons were still migrating posteriorly. The leading as well as the following neurons of one migratory pathway showed strong cGMPIR. (H) During the phase of lateral neurite branching and the formation of terminal processes on the midgut musculature, the MG neurons continued to exhibit strong cGMP-IR. These micrographs were compiled from several focal planes. Scale bars: 50 µm in A,B; 20 µm in C,D; 200 µm in E,F; 25 µm in G,H.

View larger version (18K): [in a new window]   Fig. 3. MG neuron migration under the influence of neurochemicals that affect the NO/cGMP signalling pathway. Images were drawn from individual guts that were stained with anti--tubulin antiserum. At the beginning of the experiment, MG neurons migrated not more than 40 µm on the midgut surface (start). The next drawing shows normal MG neuron migration after 24 hours incubation under control culture conditions (control). MG neuron migration was inhibited in an embryo that was exposed to 500 µM 7NI (7NI). This disruptive effect of 7NI could be rescued by the addition of 1 mM protoporphyrin IX free acid so that the MG neurons again covered the normal distance (7NI + protop.). MG neuron migration was reduced in an embryo that was exposed to 200 µM ODQ (ODQ). The inhibitory effect of ODQ was rescued by the addition of membrane permeable 8Br-cGMP (ODQ 200 µM + 8Br-cGMP). Scale bar: 200 µm.

View larger version (31K): [in a new window]   Fig. 4. Blocking of MG neuron migration by enzyme inhibitors of the NO/cGMP/PKG pathway and the cAMP/PKA cascade. Histograms show the distance migrated by the leading MG neuron on the midgut. (A) After 24 hours, normal migration of MG neurons was found in cultured control embryos (n=21). MG neuron migration was significantly reduced in the presence of 500 µM 7NI (n=21), 200 µM ODQ (n=20) and 50 µM RPcGMPS (n=21). (B) ODQ reduced MG neuron migration in a concentration-dependent manner. Normal migration of MG neurons was highly significantly inhibited in embryos that were cultured in the presence of 200 µM ODQ (control, n=21; 200 µM, n=20; 100 µM, n=20; 50 µM, n=24). (C) Histogram shows that incubation in 500 µM 7NI resulted in a significant reduction of the migratory distance (7NI; n=21) when compared with cultured control embryos (control). This disruptive effect of 7NI could be rescued by the addition of 1 mM protoporphyrin IX free acid so that the MG neurons again covered the normal distance (7NI + protoporphyrin; n=20). (D) Incubation in 200 µM ODQ resulted in a significant reduction of the migratory distance (ODQ; n=20) when compared with cultured control embryos (control; n=21). This disruptive effect of ODQ could be rescued by the addition of 8Br-cGMP such that the MG neurons again covered the normal distance (ODQ + 8Br-cGMP; n=20). (E) MG neuron migration was significantly reduced in the presence of 100 µM forskolin (n=20) and 50 µM SPcAMPS (n=21). By contrast, 50 µM RPcAMPS had no significant effect on migration (n=21) when compared with controls (control). **P<0.005; ***P<0.001.

View larger version (39K): [in a new window]   Fig. 5. F-actin staining in isolated MG neurons. (A) The control shows a representative example of the so called migratory-phenotype, with F-actin staining in the neurite and only a single fiber bundle in the soma (red). When the embryos were cultured in ODQ, the MG neurons showed a stationary-phenotype with multiple actin fiber bundles in the somata (ODQ). Co-staining with anti-HRP antiserum (green) reveals the neuronal identity of the cell (anti-HRP), whereas non-neuronal cells are unlabelled. Scale bars: 10 µm. (B) Quantitative evaluation of the actin cytoskeleton. After incubation in neurochemicals for 24 hours, a high percentage of MG neurons show the normal migratory phenotype in cultured control embryos (control). Conversely, a significantly smaller number of neurons with migratory phenotype was found in the presence of NOS inhibitor 7NI (500 µM). The disruptive effect of 7NI on the actin cytoskeleton could be rescued by the addition of 1 mM protoporphyrin IX free acid (7NI + protop.). Similarly, the number of MG neurons showing the migratory phenotype was reduced after incubation in the sGC inhibitor ODQ (200 µM). This disruptive effect of ODQ on the actin cytoskeleton organization could be rescued by the addition of 500 µM 8Br-cGMP (ODQ + 8Br-cGMP). (B) Neurochemicals that affect the cAMP/PKA pathway also had an effect on the actin organization. A significantly smaller number of neurons from embryos cultured in the AC activator forskolin (100 µM) or the PKA activator SPcAMPS (50 µM) showed the migratory phenotype as compared with controls. The PKA inhibitor RPcAMPS (50 µM) had no significant effect on the actin cytoskeleton organization. **P<0.005; ***P<0.001.

View larger version (74K): [in a new window]   Fig. 6. Time-lapse video microscopy of living MG neurons migrating on the midgut. Examples show DiO-labeled MG neurons from embryos staged between 64% and 68% E. Time intervals between the images are 20 minutes. On average, MG neurons migrated 12 µm/hour under normal conditions. To examine the effects of inhibiting sGC on individual neurons, the preparation was incubated in culture medium containing 200 µM ODQ (right). Under these conditions, there was no misrouting of the neurons and they completely stopped migrating. Scale bars: 10 µm.