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First published online 19 November 2008
doi: 10.1242/dev.026716
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University of Veterinary Medicine Hannover, Division of Cell Biology, Institute of Physiology, Bischofsholer Damm 15, D-30173 Hannover, Germany.
* Author for correspondence (e-mail: gerd.bicker{at}tiho-hannover.de)
Accepted 18 September 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Carbon monoxide, Nitric oxide, Insect nervous system, Stomatogastric, Grasshopper embryo, Cyclic GMP
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Copenhaver,
2007). In the hemimetabolous grasshopper Schistocerca,
the enteric midgut neurons of the grasshopper embryo migrate posteriorly from
caudal pockets of the ingluvial ganglia towards the foregut-midgut boundary of
the embryo (Ganfornina et al.,
1996). Subsequently, they undergo a rapid phase of migration and
move in four migratory pathways posteriorly on the midgut surface. At the
completion of migration, the enteric neurons invade the space between the four
migratory pathways and extend terminal branches on the midgut musculature
(Ganfornina et al., 1996).
Neuronal motility along the migratory pathways of the grasshopper midgut
depends crucially on the nitric oxide/cyclic GMP (NO/cGMP) signaling cascade,
including nitric oxide synthase (NOS) and the target receptor protein soluble
guanylyl cyclase (sGC) (Haase and Bicker,
2003).
NO has been reported to be a key signaling molecule during nervous system
development (Peunova et al.,
2001; Chen et al.,
2004; Bicker,
2005; Krumenacker and Murad,
2006; Godfrey et al.,
2007). In particular, the downstream acting cyclic nucleotide cGMP
is implicated as intracellular mediator of growth cone behavior both in
vertebrate and invertebrate nervous systems
(Gibbs and Truman, 1998;
Polleux et al., 2000;
Seidel and Bicker, 2000;
Song and Poo, 2001;
Van Wagenen and Rehder, 2001;
Schmidt et al., 2002;
Demyanenko et al., 2005;
Welshhans and Rehder, 2005;
Gutierrez-Mecinas et al.,
2007; Stern and Bicker,
2008).
In addition to NO, there is also increasing evidence for carbon monoxide
(CO) as another gaseous messenger of neural tissues
(Boehning and Snyder, 2003).
CO is generated by heme oxygenase enzymes (HO) during oxidative degradation of
heme to biliverdin-IX and ferrous iron
(Tenhunen et al., 1968;
Maines, 1997). Similar to NO,
CO is able to activate the cGMP-synthesizing enzyme sGC, albeit about 100-fold
less effective (Kharitonov et al.,
1995; Denninger and Marletta,
1999; Baranano and Snyder,
2001; Koesling et al.,
2004). In vertebrate nervous systems, the constitutive isoform
HO-2 is the predominant heme oxygenase
(Sun et al., 1990;
Verma et al., 1993). In the
majority of brain regions, HO-2 mRNA appears to be colocalized with that of
sGC, whereas NOS and sGC transcripts show hardly any overlap
(Verma et al., 1993). Although
NO is the major activator of sGC, CO seems to reduce cGMP levels by modulating
the effect of NO on sGC (Ingi et al.,
1996a). At the mechanistic level, CO has been suggested to mediate
long-term adaptation in amphibian olfactory receptor neurons
(Zufall and Leinders-Zufall,
1997), to serve as a neurotransmitter in
nonadrenergic/noncholinergic (NANC)-dependent smooth muscle relaxation
(Boehning and Snyder, 2003),
and to increase field potential oscillations in an invertebrate olfactory
system (Gelperin et al.,
2000). Exogenous CO application or administration of hemin
influences human neutrophil migration and platelet aggregation via cGMP
(Brüne and Ullrich, 1987;
VanUffelen et al., 1996;
Andersson et al., 2002;
Freitas et al., 2006). In the
vertebrate enteric nervous system, NOS and HO-2 are either co-expressed in a
subset of myenteric neurons, or localized in separate, but nearby, cells. NO
and CO may function here as co-neurotransmitters, with CO modulating the NO
signaling pathway (Maines,
1997; Xue et al.,
2000; Miller et al.,
2001; Colpaert et al.,
2002; Boehning and Snyder,
2003).
There are comparatively few studies about the distribution of HO enzymes
and the functions of CO as messenger molecule in invertebrate nervous systems.
Immunoreactivity to HO-2 has been described in the olfactory system of a
mollusc (Gelperin et al.,
2000) and the stomatogastric system of crayfish
(Christie et al., 2003). A HO
gene is expressed in the honeybee brain
(Watanabe et al., 2007) and
the catalytic properties of a recombinant Drosophila HO protein have
been analyzed (Zhang et al.,
2004). Unlike for NO signaling in arthropods, which could be
linked to discrete developmental processes such as cell proliferation
(Kuzin et al., 1996;
Champlin and Truman, 2000;
Benton et al., 2007), neurite
outgrowth (Seidel and Bicker,
2000), patterning of synaptic connectivity
(Ball and Truman, 1998;
Gibbs and Truman, 1998;
Wright et al., 1998) and
axonal regeneration (Stern and Bicker,
2008), no neurodevelopmental functions of CO have so far
emerged.
To investigate whether CO is a cellular messenger molecule in insect
development, we use immunochemical techniques for the localization of HO in
comparison with NOS during the formation of the grasshopper enteric nervous
system. With the concept in mind that CO may interact with NO/cGMP signaling
(Ingi et al., 1996a;
Artinian et al., 2001), we
focus on the NO-dependent cell migration of the midgut plexus neurons.
Application of enzyme substrates, chemical inhibitors, activators, messenger
releasing compounds and scavengers in whole-embryo culture implicate CO as an
intracellular messenger molecule that modulates transcellular NO signaling
during neuronal migration.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) with
additional criteria for later embryos (Ball
and Truman, 1998). All chemicals were purchased from Sigma (St
Louis, MO) unless stated otherwise.
Immunocytochemistry
All steps of immunocytochemistry were performed at room temperature and
with smooth agitation unless stated otherwise. Embryonic guts were dissected
and collected as whole-mount preparations in cooled Leibowitz 15 medium (L15,
Gibco Life Technologies, Paisley, UK). To reduce the background caused by the
yolk inside guts, some whole guts were dissected and transferred to a
Petri-dish containing a poly-D-lysine-coated coverslip with L15
culture medium. Subsequently, guts were carefully rolled over the coverslips.
During this `tissue blotting' procedure, cells on the gut surface, including
neurons of the plexus, muscle fibers and ganglia of the ENS adhered to the
coated surface, while the intact epithelium and yolk could be removed. To
ensure sufficient adherence of the nerve cells, preparations were allowed to
settle for about 30 minutes at room temperature ahead of fixation.
Preincubation for NO-dependent cGMP immunocytochemistry was carried out as
already described (De Vente et al.,
1987; Haase and Bicker,
2003) but with adding YC-1
[3-(5'-hydroxymethyl-2'-furyl)-1-benzyl Indazole, 25 µM], a
sensitizer of sGC, immediately after preparation
(Ott et al., 2004). Omitting
the NO-source from the cGMP-preincubation solution or adding sGC inhibitors,
revealed no cGMP-IR at all. All specimens were fixed in 4% paraformaldehyde
(PFA) dissolved in phosphate-buffered saline (PBS, 10 mM sodium phosphate, 150
mM NaCl, pH 7.4) overnight at 4°C. Preparations were permeabilized in 0.3%
saponin in PBS for 1 hour, rinsed in PBS containing 0.5% Triton X-100 (PBS-T)
and blocked for at least one hour in 5% normal serum/PBS-T (serum of animal in
which secondary antibody was raised). The primary antibody was diluted in
blocking solution and applied overnight at 4°C. Used primary antibodies
and concentrations were: sheep anti-cGMP (1:10,000-1:20,000)
(Tanaka et al., 1997),
monoclonal mouse anti-acetylated 
-tubulin (1:500-1:1000) and polyclonal
rabbit anti-heme oxygenase 2 (1:400-1:1000, Stressgen, Victoria, BC, Canada).
After rinsing in PBS-T, guts were exposed for at least 2 hours at room
temperature or overnight at 4°C to biotinylated (Vector, Burlingame, CA)
or AlexaFluor488-coupled (Molecular Probes, Eugene, OR) secondary antibodies
in blocking solution. Biotinylated secondary antibodies were visualized using
fluorescent streptavidin-coupled dyes (Sigma, Molecular Probes). After washing
in PBS-T and PBS, preparations were cleared in 50% glycerol (Roth, Karlsruhe,
Germany)/PBS and mounted in 90% glycerol/PBS with 4% n-propyl-gallate. Control
preparations incubated with 5% normal serum instead of primary antibodies and
subsequent detection system revealed absolutely no staining. For double
labeling, ENSs were stained first for cGMP followed by acetylated
-tubulin immunocytochemistry.
Western blotting
To obtain tissue homogenates of embryonic CNS, whole brains and ventral
nerve cords were dissected. For homogenates of the ENS, we used complete gut
tissue with the yolk removed. Tissue was dissected in cooled PBS, collected
and homogenized in ice-cold 0.3% Saponin-PBS containing 1% protease inhibitor
cocktail (HALT, Pierce, Rockford, IL). Samples of 20 (ENS) or 10 (CNS) embryos
were collected per 200 µl lysis buffer. Homogenization was carried out
using a Kontes Duall tissue grinder with PTFE pestle (Landgraf Laborsysteme,
Langenhagen, Germany). Homogenates were centrifuged for 10 minutes at 6000
g to allow a crude separation of cytosolic soluble proteins
from membrane-bound protein fractions and cellular debris. For HO-2
immunoblots, proteins were precipitated with acetone for 16-48 hours at
-20°C. Protein pellets were re-dissolved in 2x Laemmli-buffer (100
mM Tris-HCl, pH 6.8, with 4% SDS, 20% glycerol, 0.02% bromophenol blue) after
centrifugation and ethanol washing. For universal NO synthase (uNOS)
immunoblots, homogenates were used without any precipitation. Prior to
SDS-PAGE, samples were denaturated at 95°C for 3 minutes in loading buffer
(2x Laemmli buffer with 2% SDS, 10% 1 M DTT). Proteins were separated
either on 8% (NOS) or 15% (HO-2) PAGE and transferred to a PVDF-membrane
(Roth). Membranes were equilibrated in PBS, blocked for at least 1 hour at
room temperature and incubated overnight at 4°C with the antibody
dissolved in blocking solution. The following antibodies and blocking
solutions were used: polyclonal rabbit anti-uNOS (1:400, Affinity Bioreagents,
Golden, CO) in 5% low-fat milk powder (Humana, Herford, Germany) in PBS
containing 0.05% TWEEN (PBS-TW); polyclonal rabbit anti-HO-2 (1:400-1:1000),
blocked with 1% bovine serum albumin (BSA) in PBS-TW. Membranes were then
rinsed with PBS-TW and incubated with biotinylated secondary antibody in the
appropriate blocking solution for 2 hours at room temperature. After washing
with PBS-TW, bound antibodies were visualized by standard peroxidase staining
techniques using the Vectastain ABC Kit (Vector). To estimate total cell mass
in immunoblots from different tissues, some blots were stained additionally
for -tubulin. Stained membranes were dried, scanned, and after
reactivation of the membrane with methanol, the blot was probed but with
anti-acetylated -tubulin diluted 1:10,000 in PBS-TW containing 5%
low-fat milk powder.
For a specificity control of the HO-2 antiserum, purified recombinant rat HO-2 (rHO-2, Stressgen) was applied to a 15% SDS-PAGE in parallel with locust homogenates. Homogenates were divided in two aliquots and separated on the identical gel. After blotting, half of the membrane was incubated with antibody, while a pre-adsorbed antibody was applied to the other half. For pre-adsorption, recombinant rat HO-2 was added to the anti-HO-2 solution (75 µg protein/ml) and pre-incubated overnight at 4°C. Western blot analysis was repeated at least three times for each protein with homogenates from several preparations and developmental stages.
In vivo culture experiments
Embryos were staged between 60 and 65% E. An optimal stage for in vivo
chemical manipulation of cell migration on the midgut is 63% E. At this stage,
which is indicated by the first appearance of brownish pigmentation at the
tips of the antennae, midgut neurons have just started their cell migration
(Fig. 1A).
Eggs of one clutch were sterilized in 70% ethanol and dissected in sterile
L15 medium. Subsequently embryos were randomly divided into groups that were
exposed to the pharmacological compounds or the control media, respectively.
Embryos were immobilized in Sylgard embedded Petri dishes and covered with
cell culture medium, supplemented with 1% penicillin-streptomycin solution. A
small incision in the dorsal epidermis above the foregut allowed access of
pharmacological agents to the developing ENS during the in vivo culturing
period. Following an incubation for 24 hours at 30°C, guts were dissected
and prepared for anti-cGMP and anti-acetylated -tubulin double
staining.
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) using NIH ImageJ (v. 1.35-1.39). The obtained values
were normalized with respect to the mean distance of migration of the control
group. A Wilcoxon Mann-Whitney test was employed for statistical comparisons
of experimental and control groups using KyPlot (version 2.0 beta15). All
significance levels are two-sided. Bar graphs display mean
values±s.e.m. as percentage of the matched control values of each
experiment. Fig. 1B displays the mode of action of chemical agents we used to manipulate NO and CO signal transduction cascades. L-arginine and hemoglobin were predissolved in L15. Hemin and zinc protoporphyrin-IX (ZnPP-IX, Alexis, San Diego, CA) were predissolved in 0.1 M NaOH resulting in a final concentration less than 0.5% NaOH in the culture medium. 7-nitroindazole (7-NI), YC-1, tricarbonyldichlororuthenium (II) dimer (CORM II) and zinc deuteroporphyrin-IX 2,4 bis glycol (ZnBG, Alexis) were predissolved in DMSO, resulting in less than 0.5% DMSO in the culture medium.
Cell viability and cytotoxicity test
To exclude neurotoxic side effects of hemin and CO-donor applications, a
cell viability assay (Live/Dead Viability/Cytotoxicity Kit for animal cells,
Molecular Probes) was carried out. This assay allows for a clear simultaneous
discrimination between living and dead cells using two different fluorescent
probes that indicate distinct parameters of cell viability: intracellular
esterase activity and plasma membrane integrity. Immediately after in vivo
culturing, guts were dissected and the tissue was blotted on
poly-D-lysine-coated coverslips. Subsequently tissue blots were
incubated in the assay reagents for 30 minutes at room temperature in the
dark, followed by image acquisition. After counting of living and dead enteric
neurons, the percentage of living neurons was calculated.
Image acquisition and processing
Preparations were analyzed and photographed using a Zeiss Axioscope
equipped with an Axiocam3900 digital camera linked to a Zeiss image
acquisition system (Zeiss Axiovision) or a Zeiss Axiovert 200 equipped with a
Photometrics Cool Snap digital camera and associated MetaFluor Imaging
software. Confocal images of selected preparations were taken with a Leica TCS
SP2 confocal microscope using Leica LCS software. Image processing, including
arrangement, conversion to grayscale, inversion and contrast enhancement, were
carried out using Adobe Photoshop or NIH ImageJ (W. S. Rasband, ImageJ, US
National Institutes of Health, Bethesda, MD,
http://rsb.info.nih.gov/ij/).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), the
enteric neurons express NO-induced cGMP immunoreactivity
(Haase and Bicker, 2003). To
obtain a high resolution image of the cellular distribution of cGMP, we probed
tissue blots of the midgut for acetylated -tubulin and cGMP. Confocal
microscopy showed an evenly distributed cGMP-IR throughout the entire cell
bodies of enteric neurons, growth cones and the trailing neurites
(Fig. 2A,C). Neither the
underlying gut epithelium nor the developing musculature show any cGMP-IR,
whereas these tissues are clearly stained with an antibody against
-tubulin, indicative for stable microtubules
(Fig. 2B,C). Whereas filopodia
and thinner cell processes of the migratory neurons typically are lacking
immunoreactivity for acetylated -tubulin, these cell structures show
strong cGMP-IR (Fig. 2B,C).
This NO-dependent cGMP synthesis suggests a source of NO in the adjoining
tissue, that might stimulate the formation of cGMP in the enteric neurons,
including their highly motile filopodia. As cytochemical markers for NOS did
not label the motile neurons but indicated staining in the midgut epithelium
(Haase and Bicker, 2003), we
used an additional biochemical approach. Immunoblots of CNS homogenates from
65% E locust embryos revealed a protein of about 135-140 kDa that was clearly
recognized by the universal NOS antibody
(Fig. 2D, CNS). This
immunostained protein is also present in homogenates of the embryonic foregut
(FG) as well as of the midgut (MG, MG-M, midgut homogenates do also include
the adjacent hindgut) (Fig.
2D). An additional, somewhat smaller, protein of 
128 kDa was
labeled that is missing in the membrane fraction of midgut (MG-M) and is only
very faintly visible in the CNS lane by the uNOS antibody. This double band
appearance of NOS may be due to post-translational modifications, with the
smaller one representing pre-processed or degraded proteins. To normalize for
total protein content, some immunoblots were additionally marked for
-tubulin (Fig. 2D,
bottom lane). We estimate from the ratio of the loading control to the NOS
band, that the midgut contains in average a two times higher NOS concentration
than the CNS probe (midgut cytosol 0.30 and midgut membrane 0.33 versus 0.18,
n=4).
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). We could confirm that bath application of the NOS inhibitor
7-NI results in a significant reduction of enteric neuron migration
(Fig. 3A). Moreover, in vivo
incubation of 63% E embryos with the extracellular NO scavenger hemoglobin
revealed a similarly strong reduction in the migrated distance
(Fig. 3A). Most likely, NO
released by cells of the gut (Fig.
2D) serves as a transcellular regulator of neuronal cell
migration.
To test whether enteric neuron migration could be enhanced by a stimulation
of NO/cGMP signal transduction, embryos of 63 to 65% E were allowed to develop
in culture with an excess of the NOS substrate L-arginine (2 mM). The
substrate activation of NOS did not result in any significant increase of
migrated distance (Fig. 3B). NO
donors, such as sodium nitroprusside or S-nitroso-N-acetyl-l,l-penicillamine,
also failed to enhance enteric neuron migration (data not shown). Stimulation
of sGC by application of 65 µM YC-1 (Ko
et al., 1994; Evgenov et al.,
2006) appeared to slightly increase the migrated distance;
however, this result was not statistically significant
(Fig. 3B).
Carbon monoxide acts antagonistical to nitric oxide in enteric neuron migration
Heme oxygenase activity can be inhibited with low concentrations of
metalloporphyrins such as ZnBG or ZnPP-IX
(Maines, 1981;
Verma et al., 1993;
Ingi et al., 1996a;
Ingi et al., 1996b; Appelton
et al., 1999; Labbe et al.,
1999). Embryos at stages 63-65% E were exposed in culture to 5
µM ZnBG, leading to a highly significant increase of the average migrated
distance up to 131% (Fig. 3C).
Figs 4A,B illustrate that HO
inhibition by ZnBG enhances enteric neuron migration without affecting the
precision of pathfinding on the migratory routes. Using ZnPP-IX (10 µM) as
another HO inhibitor, we obtained a similar gain-of-function result
(Fig. 3C).
By stimulating the production of CO, we performed a complementary
experiment and applied 100 µM hemin as a substrate analogue for HO to the
cell culture medium. Activation of HO indeed resulted in an opposite effect,
as enteric neuron migration was significantly reduced
(Fig. 3C,
Fig. 4A,C). Moreover, exogenous
application of carbon monoxide by using the CO releasing compound CORM-II
(Motterlini et al., 2002)
reduced enteric neuron migration in a similar way to stimulation of HO enzymes
(Fig. 3C,
Fig. 4A,D). In an additional
control experiment, we tested whether the carbon monoxide exhausted compound
might account for the slowing down of enteric neuron migration. Therefore,
CORM-II was dissolved in DMSO and kept for 24-48 hours at room temperature and
used afterwards. This exhausted CORM-II (iCORM) did not affect enteric neuron
migration (Fig. 3C). Neither
hemin nor the CO-donor caused any decrease in cell viability at used (hemin)
or even higher (CORM-II) concentrations
(Fig. 3D). All of these
findings are in line with a negative regulatory role of HO activity and CO
signaling on enteric neuron migration.
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30-35 kDa
(Fig. 5A). These proteins are
comparable in size with recombinant rat HO-2 (rHO-2), labeled with the same
antibody (Fig. 5B, first two
lanes). Remarkably, anti-HO-2 labeled proteins are mainly present in the
membrane fraction of homogenates of gut and CNS
(Fig. 5A, left lanes). This is
in line with reports about an association of vertebrate HO-2 with the
endoplasmic reticulum and nuclear outer membrane
(Maines, 1988;
Ma et al., 2004). The multiple
band appearance and the slightly heavier proteins between 35 and 45 kDa that
is visible in cytosolic portions may be a result of post-translational
modifications. Diverse cleavage of the C terminus of the protein has also been
often observed during separation of HOs on SDS gels
(Ding et al., 1999). To test the specificity of the antibody against vertebrate HO-2 on invertebrate tissues, we used pre-adsorbed antibody-solution (75 µg rHO-2/ml). Pre-adsorption leads to a complete loss of labeling of the 30-35 kDa protein bands from locust homogenates (Fig. 5B, arrowhead). Additional labeled proteins around 60 kDa become visible, when rHO-2 is applied in high quantity (1 µg) to the gel (Fig. 5B, left side/HO-2). As they are not recognized by the pre-adsorbed antibody, these bands are probably due to aggregates of HO-2 (Fig. 5B, right side/HO-2). Such high protein concentrations may also account for some of the larger molecular weight protein bands in homogenates. However, as pre-adsorption abolished both the labeling of 30-35 kDa protein bands derived from recombinant HO-2 and embryonic locust, we can safely infer the presence of HO in the developing CNS and gut tissue.
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-tubulin-IR (Fig. 6D).
In summary, the results of western blotting and immunocytochemistry indicate
the presence of HO in embryonic guts and the migrating enteric neurons. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although a genetic approach would be very useful
to reveal additional downstream molecular pathways of NO and CO signaling
during insect embryonic development, the application of chemical agents at
precise time intervals is more suited to detect subtle modulatory influences
of the gaseous messengers.
NOS expression provides for a transcellular NO signal during formation of enteric midgut plexus
The uNOS antibody stains NADPH-diaphorase-positive interneurons in the
adult locust antennal lobe (Bicker,
2001) that express Ca2+/calmodulin sensitive NOS
(Müller and Bicker, 1994;
Elphick et al., 1995). In the
absence of molecular sequence data for the NOS of locusts, we used a universal
NOS antibody that recognizes a highly conserved sequence of the three
mammalian NOS isoforms and also detects arthropod NOS
(Bicker, 2001;
Bullerjahn and Pflüger,
2003; Christie et al.,
2003; Settembrini et al.,
2007). Western blotting of homogenates from embryonic fore- and
midguts revealed the presence of NOS-like 135-140 kDa proteins at the onset of
enteric neuron migration (Fig.
2D). The approximate size of NOS has been independently determined
as 135 kDa in the locust brain (Elphick et
al., 1995), between 116 and 180 kDa in the locust abdominal
nervous system (Bullerjahn and
Pflüger, 2003) and between 130 and 150 kDa in other insects
(Gibson and Nighorn, 2000;
Watanabe et al., 2007;
Settembrini et al., 2007). The
genome of Drosophila contains only a single NOS gene (Regulsky and
Tully, 1995; Enikopolov et al., 1999) and immunoblotting showed a band of the
corresponding protein at about 150 kDa
(Regulski et al., 2004). Our
results indicate highly levels of NOS proteins in the midgut, compared with
the CNS (Fig. 2D). Most likely,
this is caused by high levels of NOS expression in a substantial fraction of
the epithelial gut cells. Using quantitative PCR, NOS expression has also been
found in the midgut epithelium of Anopheles
(Akman-Anderson et al., 2007).
NADPHd-diaphorase histochemistry detected staining in a subset of the locust
embryonic midgut cells (Haase and Bicker,
2003) and hemocytes (data not shown) as potential endogenous
NO-producing cells. However, as the diaphorase reaction depends on fixation
conditions (Ott and Burrows,
1999), it may quite often not completely resolve the expression
pattern of NOS. As application of hemoglobin as an extracellular scavenger of
gaseous molecules suppressed enteric neuron migration, a transcellularly
diffusing NO signal derived from the gut cells may stimulate cGMP-mediated
enteric neuron migration. This concept is in line with the immunocytochemical
localization of cGMP in the cell bodies and filopodia after exogenous
application of NO and the sGC sensitizer YC-1
(Fig. 1B,
Fig. 2A,C)
(Haase and Bicker, 2003).
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;
Watanabe et al., 1998;
Oess et al., 2006). Therefore,
the double band appearance of NOS proteins that we observed in homogenates of
locust embryos (Fig. 2D) might
be due to post-translational modifications similar to vertebrate NOS
modifications accompanying protein membrane association.
CO is an inhibitor of enteric neuron migration
In a gain-of-function experiment we could show that inhibition of HO with
low concentrations of ZnBG and ZnPP-IX resulted in a significant acceleration
of migration on the midgut (Fig.
3C, Fig. 4A,B).
Conversely, we could significantly delay enteric neuron migration by
stimulating CO production with the HO substrate analogue hemin or with
exogenous CO application (Fig.
3C, Fig. 4A,C,D).
This confirms CO as a likely effector molecule of HO activation on the
regulation of enteric neuron migration.
Unlike to the locust, application of 10 µM ZnPP-IX to the developing
Manduca ENS caused no significant enhancement of enteric neuron
motility (Wright et al.,
1998). However, despite the common developmental origin from
neuroepithelial parts of the foregut, enteric nervous systems of insects
exhibit extensive variations in the detailed pattern of cell migration and
underlying molecular guidance cues
(Hartenstein, 1997;
Ganfornina et al., 1996;
Copenhaver, 2007). For
example, whereas in Manduca specific sets of visceral muscle bands
support migration of the enteric neurons on the midgut
(Copenhaver and Taghert, 1989;
Copenhaver et al., 1996;
Wright et al., 1999) no
morphologically distinct muscle bands can be recognized along the migratory
pathways in the grasshopper embryo
(Ganfornina et al., 1996).
Instead, the migratory neurons move parallel to the longitudinal muscle bands
directly on the surface of the midgut.
Specificity of chemical manipulations
At high concentrations, some metalloporphyrins have been reported not only
to block HOs but also to inhibit sGC or NOS activity
(Luo and Vincent, 1994;
Grundemar and Ny, 1997;
Serfass and Burstyn, 1998). An
example is the potent inhibitor of HOs ZnPP-IX
(Maines, 1981), which was
shown to cause such unspecific side effects later on. However, the low
concentrations of ZnBG (5 µM) or ZnPP-IX (10 µM) that we applied have
been demonstrated to be highly effective for the inhibition of HO enzyme
activity without influencing NOS or sGC
(Ingi et al., 1996a;
Appleton et al., 1999).
Moreover, the direction of observed motility changes, rules out an additional
inhibitory effect on sGC or NOS. As shown by
Fig. 3A, a direct inhibition of
NOS with 7-NI or sGC with ODQ (Haase and
Bicker, 2003) revealed a significant retardation in enteric neuron
migration. Thus, an additional inhibition of NOS/sGC enzymes by ZnBG or
ZnPP-IX would have slowed down neuronal migration. As the application of
metalloporphyrins caused a significant acceleration of migration
(Fig. 3C), our results provide
no evidence for unspecific side effects.
Application of the extracellular scavenger hemoglobin delayed migration (Fig. 3A). As hemoglobin binds both NO and CO, the net effect of a reduced motility can therefore be caused by a reduced concentration of both compounds in the extracellular space. Surprisingly, the reduced motility after incubation with hemoglobin matches the reduction after inhibition of NOS, whereas a lack of CO leads to an opposite effect of accelerating neuron migration (Fig. 3A,C). This result would suggest that the net effect of hemoglobin could be mainly due to scavenging extracellular NO. Moreover, the presence of the HO enzyme in enteric neurons and the lack of NOS (Fig. 2D, Fig. 6A-C) argue for an intracellular CO and transcellular NO signal transduction mechanism. However, in the absence of real concentration measurements of intra- and extracellular CO/NO levels before and after hemoglobin application, it is difficult to draw any firm conclusions. It remains a distinct possibility that CO produced in an enteric neuron may not only act intracellularly but that some of the CO escaping in the extracellular space may also downregulate the motility of neighboring cells.
Regulation of cell motility by gaseous messengers
The most straightforward functional explanation for all our reported data
is a scenario in which the gut cells provide a transcellular NO signal
(Fig. 2D,
Fig. 3A) for stimulating sGC in
the enteric midgut neurons (Fig.
2A-C). NO-induced cGMP synthesis is in turn a permissive, but
essential prerequisite for enteric neuron migration
(Haase and Bicker, 2003). An
additional CO pathway within and between the HO-positive midgut neurons
provides for an auto/paracrine signal that downregulates neuronal motility
along the chain of migrating enteric neurons
(Fig. 3C,
Fig. 4). We propose that both
gaseous messenger molecules interact via sGC to organize the timing of the
posterior directed cell migration along the midgut. As NO and CO can both bind
to sGC, but CO with less efficiency to stimulate cGMP formation
(Kharitonov et al., 1995;
Denninger and Marletta, 1999;
Baranano and Snyder, 2001;
Koesling et al., 2004), a
simple competition mechanism between the two messengers may regulate cGMP
levels in the migratory neurons (Fig.
1B). In the presence of NO and CO, part of sGC enzymes would bind
CO resulting in a suboptimal cGMP production. By decreasing CO concentrations,
an increasing number of sGC enzymes would be available for efficient NO
activation. A decrease in CO concentration by blocking HO
(Fig. 1B) would thus be
reflected by the increased cellular motility
(Fig. 3C). It is also
conceivable that CO binding causes conformational changes of sGC affecting its
efficiency (Hernandez-Viadel et al.,
2004) or perhaps induces allosteric effects arising from
interactions among sGCs or their regulatory proteins
(Ingi et al., 1996a) that
cause a downregulation of the NO stimulated cGMP production. Currently, we
cannot exclude an sGC/cGMP-independent pathway for the effect of HO/CO
signaling on enteric neuron migration, as CO can have other cellular effects
apart from modulating cGMP levels
(Boehning and Snyder, 2003;
Kim et al., 2006).
Even though the filopodial tips of migrating enteric neurons can upregulate cGMP (Fig. 2A,C), neither the chemical manipulations of NO/cGMP-nor CO-dependent signaling pathways causes misrouting of enteric neurons. Thus, a permissive effect of gaseous messengers on motility appears to be independent from growth cone steering. A quantitative evaluation of pathfinding errors (data not shown) provided no evidence for enhanced misrouting of migrating enteric neurons after the application of metalloporphyrins, hemin or CORM-II. This result is illustrated in examples of migratory pathways under the chemical manipulation of HO (Fig. 4).
To fully appreciate the role of HO/CO signaling in cell migration, it
remains insufficient to define the cellular sources and targets of CO.
Technology has yet to be developed to resolve the temporal pattern of CO
production. Although neuronal production of NO is a tightly
Ca2+/calmodulin regulated process in locusts and vertebrates
(Müller and Bicker, 1994;
Elphick et al., 1995;
Boehning and Snyder, 2003),
regulation of HO activity is thought to depend mainly on the availability of
heme. However, in response to neuronal stimulation a modest regulation of HO-2
has been demonstrated by casein kinase 2 (CK2)
(Boehning et al., 2003) and in
vitro studies have uncovered an additional mode of activation by binding of
Ca2+/calmodulin (Boehning et
al., 2004). Thus, there is increasing evidence for an
activity-dependent release of CO, one of the necessary criteria for a role as
neural messenger molecule.
Using a rather simple invertebrate model, we have uncovered an antagonistic role of CO versus NO as gaseous messenger molecules regulating nerve cell migration. As many signaling mechanisms of neuronal and cellular guidance are strikingly conserved among vertebrate and invertebrate animals, it will be interesting to examine whether CO signaling plays also a vital role during vertebrate brain development.
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