The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish
1 Department of Pathology, Lund University, Sölvegatan 25, S-221 85
Lund, Sweden
2 Department of Anatomy and Neurobiology, Boston University Medical School,
715 Albany Street R-91, Boston, MA 02118-2394, USA
* Author for correspondence (e-mail: bo.holmqvist{at}pat.lu.se)
Accepted 17 December 2003
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Summary |
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These studies indicate spatiotemporal actions by NO during embryogenesis in the formation of the central and peripheral nervous system, with possible involvement in processes such as neurogenesis, organogenesis and early physiology.
Key words: morphogenesis, zebrafish, Danio rerio, brain, retina, gut, intestine, hybridisation, in situ, development, regeneration, neuronal differentiation
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Introduction |
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NO is a free radical molecule that is formed in biological tissues from
L-arginine by three major nitric oxide synthase isoforms, nNOS,
endothelial NOS (eNOS) and inducible NOS (iNOS), using nicotinamide adenine
dinucleotide phosphate (NADPH) as a cofactor (see
Alderton et al., 2001). In
addition to NO's multifunctional properties in various normal and
pathophysiological events (see Bredt and
Snyder, 1994
; Moncada et al.,
1991
,
1998
;
Vincent, 1994
), recent studies
have also emphasized important roles for NO in early life processes
(Gouge et al., 1998
;
Jablonka-Shariff et al., 1999
;
Kuo et al., 2000
). One
proposed mechanism for the effects of NO in developmental processes is a
suppressive influence on DNA synthesis, whereby NO acts as a negative
regulator on precursor cells and thereby affects the balance of cell
proliferation, differentiation and apoptosis
(Enikolopov et al., 1999
;
Puenova et al., 2001
;
Puenova and Enikolopov, 1995
).
In the developing nervous system, studies performed in different species have
implicated NO in mechanisms such as neural differentiation, pathfinding and
synapse formation (Kuzin et al.,
2000
; Mize et al.,
1998
; Ogura et al.,
1996
; Shoham et al.,
1997
). In developing insects
(Enikolopov et al., 1999
;
Gibbs and Truman, 2000
; Kuzin
et al., 1996
,
2000
) and amphibians
(tadpoles; Puenova et al.,
2001
), NO participates in the regulation of cell proliferation,
differentiation and apoptosis, and in developing gastropods (snails),
nitrergic neurons have been demonstrated to participate in both behavioural
and physiological functions
(Serfözö and Elekes,
2002
). In mammals, the presence of NO-producing systems and
NO-mediated action in developmental processes of the CNS have preferentially
been studied during early postnatal stages (see
Mize et al., 1998
). In
different species, nNOS or nNOS-like isoforms may be the major source of
NO-mediated action in developmental and plastic processes
(Mize et al., 1998
;
Puenova et al., 2001
),
including in restricted brain areas with ongoing neurogenesis and neural
plasticity in adult mammals (Islam et al.,
1998
; Moreno-Lopez et al.,
2000
). In lower vertebrates such as teleosts, NO has been
emphasised to play a versatile role in the development of the central nervous
system during both embryonic and post-embryonic life stages
(Devadas et al., 2001
;
Fritsche et al., 2000
;
Gibbs et al., 2001
,
Gibbs et al., 2001
;
Ribera et al., 1998
). In the
brain of adult zebrafish, nNOS mRNA-expressing populations are closely
associated with the proliferation zones
(Holmqvist et al., 2000a
) that
generate new cells throughout life (see
Ekström et al., 2001
;
Wulliman and Knipp, 2000
). A
role for NO in cell proliferation zones of different brain areas was recently
demonstrated in tadpoles (Puenova et al.,
2001
), and has been indicated in more restricted ongoing
neurogenesis of the subventricular zone in adult mammals
(Islam et al., 1998
;
Moreno-Lopez et al.,
2000
).
All three major NOS isoforms are expressed during early development. The
differentiated expression of NOS isoforms in certain tissues at different
developmental stages indicates that temporal and spatial NO-mediated
activities may be regulated by different NOS-producing systems (see
Alderton et al., 2001;
Eliasson et al., 1997
;
Lee et al., 1997
;
Wang et al., 1999
,
Wang et al., 1999
). In the
developing brain, NOS enzyme activity, NOS proteins and mRNAs of the NOS
isoforms have been detected in different mammalian species; however, few
species have been studied in detail, and there has been little discrimination
between the specific NOS isoforms (see
Judas et al., 1999
). To date,
detailed analysis of NOS systems during early embryonic development have
mainly been limited to the presence of the nNOS protein in the brain of rat
and mouse. In teleosts, NOS proteins and their activity have been
characterized, and the molecular identity demonstrated for nNOS and iNOS
(Cox et al., 2001
;
Holmqvist et al., 2000a
;
Øyan et al., 2000
;
Saeij et al., 2000
). In
zebrafish and salmon, preliminary studies have shown an early expression of
nNOS mRNA in the CNS (Holmqvist et al.,
1998
,
2000b
). In adult teleosts,
peripheral organs contain nNOS systems homologous to mammals (see
Brüning et al., 1996
),
whereas the NOS isoform identities of different developing NADPHd active
peripheral systems (Villani,
1999b
) are unknown. Spatial documentation of specific NOS mRNA
expression in a whole organ has been reported for nNOS in brains of adult rat
(Iwase et al., 1998
) and
zebrafish (Holmqvist et al.,
2000a
).
Guanylate cyclase is proposed to be the major target for NO, causing an
increase in intracellular guanosine 3',5' cyclic monophosphate
(cGMP), a second messenger affecting multiple molecular targets (see
Denninger and Marletta, 1999;
McDonald and Murad, 1996
).
Data accumulated so far suggest that NO and cGMP together may play an
important role in the development of specific pathways in the CNS and
peripheral nervous system (PNS) of both vertebrates and invertebrates
(Gibbs et al., 2001
,
Gibbs et al., 2001
;
Gibbs and Truman, 2000
;
Giulli et al., 1994
;
Serfözö and Elekes,
2002
).
NO is indicated to be an important factor in early developmental processes throughout the vertebrate phylogeny. However, little is known about the detailed ontogeny of NOS-isoforms during vertebrate embryogenesis. We therefore investigated the morphological basis for putative NO-mediated actions derived from nNOS during early development of the zebrafish. The spatiotemporal expression of nNOS mRNA was investigated in the whole developing body using in situ hybridisation techniques, and was related to the neural differentiation pattern and temporal cGMP expression.
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Materials and methods |
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For in situ hybridisation, cDNA constituting the 621 bpgene
sequence encoding zebrafish nNOS mRNA (GenBank, accession number AF219519) was
used to make RNA probes (Holmqvist et al.,
2000a). Antisense and sense probes were made from cDNA inserted
into pGEM-T Easy vectors (Promega, Madison, USA) and linearized with BSP 120I
(anti-sense) or SalI (sense), respectively. Digoxigenin (DIG)
labelling of probes was performed using T7 and SP6 RNA polymerase,
respectively, according to the manufacturer's instructions (Boehringer
Mannheim, Germany). Whole mounts from life stages that achieved pigmentation
were treated with hydrogen peroxide (0.1% in methanol) for 13 h. Whole
mounts were permeabilized with Triton X-100 (1% in PBS) for 2472 h,
depending on their developmental stage. Whole mounts and sections were
postfixed in 4% paraformaldehyde in PBS, and then further permeabilized with
proteinase-K (0.25 mg ml1, 510 min at room
temperature). After immersion in fixative (4% paraformaldehyde for 10 min) and
treatment with acetic anhydride (0.25% for 10 min), whole mounts and sections
were rinsed in 5x sodium citrate buffer (SSC) and incubated for
13 h at room temperature in hybridisation buffer [50% formamide +
5x SSC + 5x Denhardt's solution (Sigma) + 250 µg
ml1 MRE 600 tRNA (Roche, Darmstadt, Germany) + 500 µg
ml1 denatured and sheared salmon testes DNA (Sigma)]. For
cryosections, 10% dextran sulphate was added to the hybridisation buffer.
Hybridisation with 600800 ng ml1 probe was performed
in hybridisation buffer for 16 h at 65°C. Post-hybridisation rinses were
performed in 5x SSC for 2x 15 min or 30 min at room temperature,
in 3.5x SSC containing 30% formamide for 30 min at 65°C, in
0.2x SSC for 2x 30 min at 65°C, and in 0.2x SSC for
2x 15 min or 2x 5 min at room temperature. Visualization of
hybridised transcripts was performed via sequential incubation with a
goat anti-DIG, alkaline phosphatase-conjugated antibody for 16 h at 8°C
(1:2000; Roche) and alkaline phosphatase reaction solution containing 3.4
µl ml1 nitro-blue tetrazolium, 3.5 µl
ml1 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche) and
0.001 mol l1 Levamisole (Sigma). The reaction was performed
for 632 h at room temperature, and was sometimes continued up to 72 h
at room temperature or at 8°C. The reaction was stopped in Tris-EDTA (TE,
0.01 mol l1). Whole mounts were immersed in TE containing
50% glycerol and mounted between coverslips. Cryosections were mounted
directly in Kaiser's glycerol gelatin (Merck, Penzberg, Germany), or were
dehydrated in an alcohol series ending with xylol, and mounted in Histomount
(Histolab, Gothenburg, Sweden).
For correlation of nNOS mRNA expression with the general neuronal
differentiation pattern, parallel sections to those used for in situ
hybridisation were labelled with monoclonal mouse antibodies against
acetylated -tubulin (AT; Incstar USA, diluted 1:1000), which
specifically detects newly differentiated neuronal structures in the zebrafish
(Chitnis and Kuwada, 1990
;
Ross et al., 1992
;
Wilson et al., 1990
). Sections
were incubated in the AT antiserum for 4872 h at 8°C, and then in
swine anti-mouse IgG antiserum (1:50; DAKO, Denmark) and mouse peroxidase
anti-peroxidase (PAP, 1:50; DAKO) for 30 min each at room temperature). Tissue
sections were then incubated with 3,3'-diaminobenzidine
tetrahydrochloride (DAB; 0.010.05%) containing
H2O2 (0.0125%) and NiSO4 (0.025%) in Tris-HCl
(0.1 mol l1, pH 7.6) for 510 min.
Sections and whole mounts were analyzed using a light microscope equipped with interference Nomarski optics (Olympus AX60, Tokyo, Japan), and digital images were collected with a digital camera (Olympus DP50-CU). Images were corrected for brightness, contrast and colour balance, and were mounted as plates using Adobe Photoshop (version 5.0 for Macintosh, Apple).
For cGMP analyses, 60 fertilized eggs per sample (of wild type used for nNOS studies, or the Tübingen strain) were sampled at 8, 14, 20, 24, 30, 34, 40 and 55 h.p.f. (these time points were also used for nNOS mRNA in situ hybridisation). Eggs were placed in a 1 ml Eppendorf tube, the medium removed, and the tube immediately placed in liquid nitrogen. Samples were stored at 80°C until the extraction procedure. As controls for cGMP levels present in the egg yolk soon after fertilization, egg samples (Tübingen strain only) were also collected at the 12 cell stage and processed in the same way.
Prior to the cGMP assay, frozen tissue was homogenized and sonicated in cold 6% trichloroacetic acid to give 10% w/v homogenate. The sample was then centrifuged at 2000 g for 15 min at 4°C, and the supernatant was then recovered and washed 4x with 5 volumes of water-saturated diethyl ether. The aqueous extract was dried in a vacuum drier (Savant, Newington, USA) and then reconstituted in assay buffer. cGMP concentrations in the embryos were measured in duplicate using 125I-cGMP radio-immunoassay kits (Amersham International, England), according to a standard acetylation protocol. In addition to the standard curve, standard cyclic nucleotide concentrations were repeatedly measured throughout the assay procedure (two different standards after every four duplicate samples) in order to ensure the stable performance of the assay. The intra-assay coefficient of variation was 4.1%. All the samples collected were analyzed for cGMP concentrations within the same extraction and assay procedure. The data were processed for statistical evaluations using an unpaired Student's t-test.
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Results |
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Temporal and spatial expression of nNOS mRNA transcripts in the CNS
Expression of nNOS mRNA transcripts was first detected in the forebrain at
19 h.p.f., after which the labelling intensity increased, both in terms of the
number of expressing cells and their anatomical distribution. The first
labelled cells (Fig. 2A,B) were
located bilaterally in the ventral forebrain close to the neuroepithelium in
the most ventrolateral position adjacent to the eye primordium, i.e.
corresponding to the ventrorostral cell cluster (vrc). Between 22 and 24
h.p.f.,additional strongly labelled cells appeared in the vrc, constituting
around 58 cell bodies at these stages
(Fig. 2C,D).
At this stage, AT-IR structures, perikarya and axonal projections, had increased significantly (Fig. 2HJ).
Between 26 and 30 h.p.f., the number of strongly labelled nNOS mRNA-expressing cells in vrc increased. At this time, additional labelled cells appeared more caudal, in a position corresponding to the ventrocaudal cell cluster (vcc; Fig. 2E).
At 34 h.p.f., relatively weak labelling of nNOS mRNA transcripts was also visualized in the dorsorostral embryonic cell cluster (drc), in hindbrain cell clusters (hc) and in the medulla (Fig. 2F,G). Between 19 and 34 h.p.f., nNOS mRNA-expressing cells had appeared in different cell populations in vrc, drc, vcc, hc and in the medulla, preceded by AT-IR populations (Fig. 2HJ).
Between 40 and 55 h.p.f., nNOS mRNA-expressing populations increased most
significantly with respect to the number of labelled cell bodies, a wide
distribution in all major brain areas, and to labelling intensity of most
populations (Figs 3,
4). At 55 h.p.f., several cell
populations were intensely labelled and were distributed in all major parts of
the brain. nNOS mRNA-expressing populations in areas that contained AT-IR
cells appeared to be nNOS subpopulations of not yet differentiated
(presumptive) brain nuclei, expressing nNOS in the adult brain
(Holmqvist et al., 2000a),
which have been anatomically defined in the adult brain
(Wulliman et al., 1996
). In
addition, large nNOS mRNA-expressing populations were present in areas that
did not have any AT-IR perikarya (Figs
3,
4), distributed along the
proliferation zones as described in larval zebrafish
(Wulliman and Knipp, 2000
).
AT-IR neuronal projections, axons and fine fibre arborisations had increased
significantly at this time (Fig.
3A,B). In the telencephalon, a large nNOS mRNA-expressing cell
population appeared in the central area (Figs
3A,
4A), of which a smaller portion
reached into the forming rostral thalamic portion of the diencephalon. In the
ventrorostral diencephalon, relatively small to large cell populations
expressing nNOS mRNA were located in the presumptive preoptic (and
suprachiasmatic) and rostral thalamic area
(Fig. 4C). A distinct nNOS cell
cluster was present in the dorsal diencephalon, located in the presumptive
pretectal area (Figs 3A,
4E). Relatively intensely
labelled nNOS cell populations were located in the presumptive posterior
tuberal, caudal and lateral portions of the forming hypothalamus (Figs
3A,
4E). A large cell cluster was
located dorsal to the hindbrain, in the mesencephalon, on the border between
the caudal portion of the optic tectum and corpus cerebelli (Figs
3A,
4E). Intensely labelled cells
were located along the ventral spinal cord close to the central canal
(Fig. 4F,H),coinciding with the
extensive AT-IR neuronal network (Fig.
4G). Scattered nNOS mRNA-expressing cells were present in the
central rhombencephalon, in areas corresponding to the facial and vagus lobes.
In the brain, most nNOS-expressing populations present in adults
(Guido et al., 1997
) had
appeared at 55 h.p.f., with no noticeable increase observed at 72 h.p.f.
In the retina, nNOS mRNA transcripts were expressed first between 4555 h.p.f., with no noticeable increase in larva, as represented by a few weakly labelled cells located in the morphologically undifferentiated inner nuclear layer (Fig. 4I).
Temporal and spatial expression of nNOS mRNA transcripts in peripheral organs
In peripheral organs (Figs
5,
6), nNOS mRNA expression was
first detected in the skin, at 20 h.p.f.,predominantly in the posterior two
thirds of the animal (Fig.
5A,B). The labelling was restricted to epithelial cells
(Fig. 5C,D). Expression in the
skin decreased during embryonic development, and only a few cells with
relatively low labelling intensity were detected at 55 h.p.f. By 72 h.p.f.
there was no labelling in the skin.
In body organs, nNOS expression was first detected at 55 h.p.f. and was associated with the forming alimentary tract (Fig. 5F). There was a significant increase in nNOS expression just after hatching, and at 72 h.p.f. widespread nNOS-expressing cell populations were detected in the vicinity of the presumptive swimbladder, gut and nephritic ducts (Figs 5GI, 6AG). Rostrally, nNOS-expressing cells were located bilaterally in the mesenchyme of the swim bladder and in the dorsolateral portion of the gut (Fig. 5GI). Larger populations of nNOS-expressing cells associated with the swim bladder were preferentially detected in the caudal portion (Fig. 6A,B). Larger clusters of nNOS-expressing cells were located at the rostral level of the alimentary tract (Fig. 6A,C,D), in presumptive enteric ganglia. More caudal nNOS-expressing populations were fewer (Fig. 6E), and cells located in the mesenchyme were evenly distributed uniformly throughout the length of the alimentary tract and nephritic duct (Fig. 6G).
Temporal cGMP expression
The production of cGMP (Fig.
7) at the early stages of zebrafish development (8 h.p.f.)
corresponded to that in 12 cell eggs (0.81.2 fmol/egg), and was
thus considered to be of extra-embryonic origin. The same temporal patterns
and absolute levels of cGMP production were observed in the two wild-type
strains of zebrafish used, both strains showing a correlated increase in cGMP
level with age. Distinct temporal changes in cGMP levels during zebrafish
embryogenesis were characterized by the rapid raise in cGMP levels between 20
and 24 h.p.f. (0.47 fmol h1) and between 40 and 55
h.p.f. (
0.38 fmol h1; P<0.05), and by the
lack of significant and slow increase in cGMP levels between 8 and 20
h.p.f.(
0.09 fmol h1) and 2440 h.p.f.
(
0.03 fmol h1).
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Discussion |
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Methodological considerations and nNOS/cGMP activity
To our knowledge, in embryonic teleost species NOS has so far only been
detected using NADPHd enzyme histochemical techniques (Villani,
1999a,b
).
The specific detection, hybridisation and visualization of zebrafish nNOS mRNA
in embryonic tissue by the anti-sense probe used and in situ
hybridisation technique is demonstrated by the lack of labelling using the
sense probe, the stringent hybridisation conditions used, and the restricted
labelling of the cytoplasm (see Fig.
2D,E,G), which comply with that shown previously in tissue from
adults (Holmqvist et al.,
2000a
). Importantly, whole-mount preparations could not be used
since whole mounts provided a lower signal of nNOS mRNA expression, which did
not detect initial expression in newly differentiated cells within the embryo,
and also the pre- and post-treatment (de-pigmentation and clearing) of whole
mounts diminished detection of transcripts in the skin (see Figs
2,
5). A corresponding, relatively
low expression of new nNOS cells in embryo was previously noted in cells
located in association with the brain proliferation zones in adult zebrafish
(Holmqvist et al., 2000a
).
Thus, for the detection of nNOS mRNA expression, cryosections yielded better
preservation, detection and visualization of transcripts, which together with
the higher morphological resolution on microscopical analysis, provided
reliable and detailed spatial, cellular and anatomical analysis of the
expression.
The initial nNOS expression appeared in areas with newly differentiated
neuronal populations, previously indicated to be NADPHd-positive in another
teleost species (Villani,
1999a,b
).
At late embryonic stages, nNOS mRNA-expressing cells formed distinct
populations in differentiating presumptive brain nuclei, possessing nNOS
protein and NADPHd activity in adult zebrafish
(Holmqvist et al., 2000a
), and
in peripheral clusters and ganglia identified as nNOS immunoreactive in
another adult teleost species (Brüning
et al., 1996
). The early nNOS mRNA-expressing cells may thus
comprise both mature and early differentiating neurons (see below). The lack
of nNOS mRNA expression in areas that are NADPHd positive or NOS
immunoreactive in adults, such as the olfactory system, brain, retina and
pineal organ, agrees with previous indications of as-yet-unknown NOS isoforms
in these systems (Holmqvist et al.
1994
,
2000a
;
Östholm et al., 1994
;
Shin et al., 2000
). Whether
teleost iNOS isoforms, possessing corresponding molecular structure and
induced expression to those in mammals
(Saeij et al., 2000
), are
expressed during development is not known. The zebrafish nNOS mRNA fragment
detected here may also be part of an alternatively spliced nNOS mRNA variant
(see Eliasson et al., 1997
;
Lee et al., 1997
;
Wang et al., 1999
,
Wang et al., 1999
), indicated
previously in a teleost species
(Øyan et al., 2000
). In
mammals, spliced nNOS mRNA variants have been shown to participate in the
differentiated developmental pattern
(Eliasson et al., 1997
;
Lee et al., 1997
;
Northington et al., 1996
;
Oermann et al., 1999
). Also,
gene duplication (see Van de Peer et al.,
2002
) may be considered for zebrafish nNOS. Further investigations
are needed to elucidate whether a specific nNOS isoform or splice variant is
preferentially engaged in the developmental processes.
The 621 bp fragment of zebrafish nNOS mRNA detected in this study has a
relatively close homology in sequence identities/similarities with the
corresponding region of nNOS in mammals. It corresponds to positions that hold
the conserved calmodulin and monoflavin binding sites, designating its
NO-producing character, and thus its functional capacity (see
Holmqvist et al., 2000a). The
spatial distribution of NOS activity by the identified nNOS systems is
supported by the corresponding cell populations expressing nNOS mRNA in
embryonic zebrafish and NOS-like activity (i.e. NADPHd activity) in embryonic
Tilapia (Villani,
1999a
,b
).
The spatial expression of nNOS mRNA, together with the temporal correlation
between the pattern of nNOS mRNA expression and cGMP levels (Figs
1,
7), may support previously
reported NO-cGMP action in developmental processes
(Giulli et al., 1994
;
Gibbs et al., 2001
,
Gibbs et al., 2001
;
Gibbs and Truman, 2000
;
Kuzin et al., 2000
). Although
guanylyl cyclase may be the major target for NO, however, NO has other
molecular targets, and the ability to alter gene expression at different
levels and via modifications of gene products (Bogdan et al., 2001).
Furthermore, cGMP signalling is involved as a second messenger in multiple
systems that do not involve NO (Denninger
and Marletta, 1999
; McDonald
and Murad, 1996
), which in our measurements may constitute an
unknown portion of the whole body cGMP (see also below).
Ontogeny of nNOS, and temporal correlations with cGMP
The initial expression of nNOS mRNA in the brain of the zebrafish follows
the formation of specific neuronal populations, the vrc and vcc embryonic
clusters, and corresponds to the first neurotransmitter differentiation in
these cell clusters. The initial neuronal differentiation in the zebrafish
development (see Kimmel et al.,
1995) begins from cellular precursors in the basal plate at
1012 h.p.f., just prior to the completion of the neural tube. The
differentiation of the specific embryonic neuronal cell clusters and axonal
scaffolds occurs around 1618 h.p.f., comprising the primary cell
clusters in the brain termed the drc, vrc, vcc, hindbrain cell cluster, the
epiphyseal and pituitary cell clusters
(Ross et al., 1992
). The
embryonic cell clusters contain the first transmitter phenotypic cells, and
the vrc and vcc in zebrafish comprise subpopulations of cell clusters holding
different primary transmitter differentiated cells, i.e. catecholaminergic,
serotonergic and gamma amino butyric acid expressing (GABA) cells
(Doldan et al., 1999
;
Ellingsen et al., 1998
;
Holzschuh et al., 2001
).
GABAergic cells are part of all embryonic cell clusters from an early stage,
whereas nNOS and the primary catecholaminergic and serotoninergic cells are
initially restricted to the vrc and vcc.
The corresponding temporal and spatial patterns for NADPHd activity in
developing Tilapia sp. (Villani,
1999a) and nNOS mRNA expression in developing zebrafish, stress a
common differentiation of nNOS cells and formation pattern of homologous nNOS
systems in the brain of teleosts. The nNOS vrc cells identified in the
zebrafish correspond to some of the first NADPHd positive cells, described in
the diencephalon of Tilapia sp. (at 20 h.p.f.). During subsequent
development, other nNOS mRNA-expressing cell populations corresponding to
NADPHd positive populations in Tilapia appear in a similar sequence
in brain areas such as the telencephalon, hypothalamus and hindbrain,
octavolateral and vagus region, and in the ventral spinal cord. At later life
stages, correlating nNOS mRNA-expressing and NADPHd positive cell populations
are those in the differentiating optic tectum and within the cerebellum.
Species differences in the labelling pattern of nNOS mRNA expression and
NADPHd labelling, such as in the olfactory placodes and parts of the
hindbrain, may be due to non-specific NADPHd labelling of other NOS-like
enzymes (as discussed above).
In the developing brain, data concerning NOS enzyme activity, NOS proteins
or mRNAs of the NOS isoforms have been reported in different mammalian species
(Derer and Derer, 1993;
Gorbatyuk et al., 1997
;
Keilhoff et al., 1996
;
Kimura et al., 1999
;
Lizasoain et al., 1996
;
Northington et al., 1996
;
Oerman et al., 1999; Takemura et al.,
1996
; Terada et al.,
1996
,
2001
;
Töpel et al., 1998
;
Wang et al., 1998
), including
human (Downen et al., 1999
;
Ohyu and Takashima, 1998
;
Yan and Ribak, 1997
). The
detailed early ontogeny of specific nNOS immunoreactive systems has been
reported in the rat brain (Terada et al.,
1996
). Together with recent preliminary data from combined
immunocytochemical, in situ hybridisation and NADPHd histochemical
studies in mouse (Holmqvist et al.,
2001
), these studies emphasize that the nNOS expression in rodents
starts in brain regions preceding the forming hypothalamus and pons. This
corresponds to the distribution of the initial nNOS populations in the
zebrafish vrc and vcc, which will form preoptic/hypothalamic regions, and in
rostral hc, which will form the rhombencephalon. In addition, in spite of the
temporal differences between rodents and zebrafish, corresponding nNOS
populations appearing during embryonic and postnatal development are present
in homologous brain regions, such as the telencephalon, thalamus,
collicular/tectal regions, cerebellum and spinal cord. In the retina of
embryonic zebrafish, relatively few and weakly labelled nNOS mRNA-expressing
cells were detected just prior to hatching, and were located in the
presumptive inner nuclear layer. In the retina of Tilapia, the first
NOS active (NADPHd positive) cell bodies appear at a similar developmental
stage. Correspondingly, in the rat, the first nNOS immunoreactive cells appear
in the inner neuroblast layer, at postnatal day 5
(Kim et al., 2000
). In late
zebrafish embryos, in addition to the nNOS populations in central brain areas,
presumptive nuclei, nNOS populations are present in the brain regions
associated with the proliferation zones
(Ekström et al., 2001
;
Wulliman and Knipp, 2000
),
shown in adult zebrafish to possess retained nNOS expression
(Holmqvist et al., 2000a
). The
close morphological relation of nNOS or nNOS-like enzymes with proliferation
zones has been noted in different brain areas of tadpoles
(Puenova et al., 2001
), and in
the more restricted brain regions exhibiting ongoing neurogenesis in adult
mammals (Islam et al., 1998
;
Moreno-Lopez et al., 2000
).
Thus, similarities in the spatial formation of specific nNOS systems in the
brain are indicated in vertebrate phylogeny, including a retained expression
throughout life in restricted regions.
The nNOS expression in peripheral organs also followed a specific
spatiotemporal pattern in developing zebrafish. Similarly, temporal
differences in expression of NOS isoforms, including nNOS, have been noted in
different tissues during development of mammals, reflecting involvement by
specific NOS isoforms or splice variants in organogenesis
(Eliasson et al., 1997;
Lee et al., 1997
;
Northington et al., 1996
;
Oermann et al., 1999
). In
developing zebrafish, nNOS mRNA expression was present transiently in skin
epithelial cells, from 20 h.p.f. and until just after hatching. NO produced by
constitutive NOS plays a role in growth and remodelling of the skin, and
NO-mediated pathological conditions are preferentially reported to be related
to iNOS and eNOS expression (Dippel et
al., 1994
; Stallmeyer et al.,
2002
). In body organs of the zebrafish, we found that the initial
expression was associated with the forming alimentary tract. The onset of nNOS
expression in peripheral organs at hatching may also contribute to the rapid
increase in cGMP expression levels recorded at this time. After hatching there
was an increase in number of nNOS cells and presumptive neurons located in
close vicinity to the swim bladder, in enteric ganglia, and in the mesenchyme
along the alimentary tract and nephritic duct. These peripheral nNOS
mRNA-expressing cell populations in zebrafish embryo are reported as NADPHd
active in developing Tilapia
(Villani, 1999b
).
Corresponding populations are both NADPHd active and NOS immunoreactive in
adult goldfish (Brüning et al.,
1996
), indicating expression by identified nNOS populations in
peripheral organs through adulthood. Peripheral organs lacking nNOS mRNA
expression but with reported NADPHd activity include the olfactory placodes,
neuromasts, otic vesicle during development, and the sensory vagal and
glossopharyngeal ganglia in adults. NOS immunoreactive but nNOS mRNA-lacking
cells include intracardiac cells, previously indicated in adult teleosts
(Brüning et al., 1996
).
Further studies of later developmental stages in zebrafish, after 72 h.p.f.,
are needed to elucidate the developmental pattern of specific nNOS in
indicated nitrergic sensory systems of peripheral organs, and/or whether they
possess a low (or undetectable) expression at the stages studied here.
Early expressed nNOS may be involved in a broad range of functions
via NO-mediated actions. The participation of NO in different
cellular processes has been documented throughout the whole animal phylogeny,
indicating its influence on different cellular processes such as mitosis and
apoptosis, neuronal pathfinding, refinement and maturation of neuronal
circuits (see Mize et al.,
1998; Moncada et al.,
1998
). In invertebrates, NO appears to be central for
morphogenesis and neurogenesis during early development, as well as for early
behavior and physiology (Enikolopov et
al., 1999
; Serfözö
and Elekes, 2002
). Corresponding roles for NO in developmental
processes in lower vertebrates are supported by recent experimental data on NO
manipulation in tadpoles (Puenova et al.,
2001
). The effects of NO can be widely distributed well beyond the
site of its origin, and beyond the classical neuronal targets, due to its
diffusive properties, thereby reaching various cellular and molecular targets.
The ontogeny of nNOS expression in zebrafish leads us to propose that NO,
produced by nNOS systems specifically, may participate in early physiology as
well as in a spatiotemporal pattern in developmental processes of
different body organs, including brain, eyes, gut, alimentary tracts and the
skin.
The influence of NO on cGMP activity related to developmental processes is
one pathway for early NO-mediated action. The coincident temporal development
of these systems in developing zebrafish, i.e. the cGMP levels accompanying
the pattern of nNOS expression (see Figs
1,
7), support this. The timing
was shown by the initial nNOS mRNA expression and the high increase in number
of nNOS-positive cells between 19 h.p.f. and 26 h.p.f., which was accompanied
by an initial increase in cGMP production (4.8% per hour) and a subsequent
major surge in cGMP expression (19% per hour), respectively. Furthermore, the
slow increase in the number of nNOS-expressing cells between 24 h.p.f. and 40
h.p.f. was accompanied by a low rise in cGMP production (0.8% per hour),
whereas the dramatic increase of nNOS-expressing cells between 40 h.p.f. and
55 h.p.f. was accompanied by a significant increase in cGMP production (8.5%
per hour). In the zebrafish, NO-cGMP actions influence the floor plate
proliferation in the spinal cord, proposed to be mediated by NADPHd active
fibres present at 2448 h.p.f.
(Gibbs et al., 2001,
Gibbs et al., 2001
). The
spatial expression of nNOS mRNA indicates that early spinal NO-mediated cGMP
action initially originates from fibres developing from the nNOS populations
in the vrc and/or vcc, whereas at later developmental stages NO-mediated cGMP
actions may occur via the nNOS-expressing cell populations located in
hc and/or in local cells in the spinal cord. NO-cGMP systems have been found
to play an essential role in the development of the visual system in
Drosophila (Gibbs and Truman,
2000
; Gibbs et al.,
2001
, Gibbs et al.,
2001
; Kuzin et al.,
2000
) and in the maturation of central visual circuits in mammals
(Giulli et al., 1994
). In
teleosts, different NOS-like isoforms
(Östholm et al., 1994
;
Shin et al., 2000
) and
guanylyl cyclase forms (Hisatomi et al.,
1999
; Seimiya et al.,
1997
) are present in the retina and photosensory pineal organ of
teleosts and may participate in NO-cGMP functions, including axogenesis and
synaptogenesis (Devadas et al.,
2001
; Villani,
1999a
), or photoreceptor light/dark adaptation
(Angotzi et al., 2002
;
Zemel et al., 1996
). The
detailed spatial relationship between the actual nNOS enzyme activity (or NO)
and cGMP-expressing target cells needs to be elucidated to determine the
cGMP-mediated functional role of the specific nNOS mRNA-expressing populations
identified in the different body organs.
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