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The ontogenic transcription of complement component C3
and Apolipoprotein A-I tRNA in Atlantic cod
(Gadus morhua L.)—a role in development and homeostasis?
Sigrun Langea,*, Alister W. Doddsb, Sigrıdur Gudmundsdottira,Slavko H. Bambira, Bergljot Magnadottira
aInstitute for Experimental Pathology, University of Iceland, Keldur v. Vesturlandsveg, Reykjavik IS-112, IcelandbMRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, UK
Received 8 November 2004; revised 15 March 2005; accepted 21 March 2005
The complement system is important both in the innate and adaptive immune response, with C3 as the central protein of all
three activation pathways. Apolipoprotein A-I (ApoLP A-I), a high-density lipoprotein (HDL), has been shown to have a
regulatory role in the complement system by inhibiting the formation of the membrane attack complex (MAC). Complement
has been associated with apoptotic functions, which are important in the immune response and are involved in organ formation
and homeostasis.
mRNA probes for cod C3 and ApoLP A-I were synthesized and in situ hybridisation used to monitor the ontogenic
development of cod from fertilised eggs until 57 days after hatching. Both C3 and ApoLP A-I transcription was detected in the
central nervous system (CNS), eye, kidney, liver, muscle, intestines, skin and chondrocytes at different stages of development.
Using TUNEL staining, apoptotic cells were identified within the same areas from 4 to 57 days posthatching.
The present findings may suggest a role for C3 and ApoLP A-I during larval development and a possible role in the
homeostasis of various organs in cod.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Complement; C3; Apolipoprotein A-I; Cod (Gadus morhua L.); Ontogeny; Embryo; Development; Apoptosis
1. Introduction
The complement system is an important element of
both the innate and adaptive immune system and is
activated through any of three pathways: the antibody-
dependentclassicalpathway, theantibody-independent
0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2005.03.009
* Corresponding author. Tel./fax: C354 5855100.
E-mail address: [email protected] (S. Lange).
alternative pathway, and the lectin pathway triggered
by the interaction of mannose-binding lectin (MBL)
or ficolins with polysaccharides [1]. Complement
consists of a group of about 30 serum proteins that
cooperate with other defence mechanisms and is in the
front line of immune defence against invading
pathogens and in clearance of potentially damaging
debris and necrotic or apoptotic cells [2]. C3 is the
central complement component. It interacts with
Developmental and Comparative Immunology xx (2005) 1–13
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many proteins, including some that participate in or
control cell adhesion and cell-to-cell communication
[3]. C3 isolated from cod serum was found to be a
two-chain (a-chain, 115 kDa; b-chain 74 kDa) glyco-
protein with an intrachain thioester bond in the a-
chain [4], similar to mammalian C3, which through its
thioester bond can covalently bind to target cells [5].
In mammals, C3 is primarily synthesised in the liver
but it has been shown that other cells and tissues, such
as monocyte/macrophages, fibroblasts, endothelial
cells, leukocytes, cells of the CNS and cells of the
renal glomerulus also produce complement com-
ponents [6]. C3 has been detected at the protein
level in various organs at different stages of cod and
halibut larval development, including cells of the
CNS, liver, eye, chondrocytes, intestines and kidney
[7,8]. The local synthesis of C3 and other complement
components in tissues other than the liver, may play
an important role in local inflammatory processes [9],
tissue remodelling [10,11] and normal reproduction
[12]. Complement-regulated pathways interact with
other signalling networks and have been shown to
influence the outcome of complex developmental
processes, such as limb regeneration in lower
vertebrates and organ regeneration in mammals [13]
and have recently been shown to be involved in stem
cell differentiation during hematopoietic development
[14]. The complement system is also involved in
apoptotic processes, by opsonising apoptotic cells and
recruiting phagocytes. Apoptotic cells may also
activate the alternative pathway directly, resulting in
C3 deposition and activation of the terminal pathway
[2,15,16]. The clearance of apoptotic cells is essential
for the prevention of an inflammatory response and
apoptosis regulates cell numbers and their fate in
embryogenesis and tissue remodelling, which is
important in development and in the maintenance of
normal tissue homeostasis [2,17,18].
ApoLP A-I is the main protein component of the
high-density lipoproteins (HDL), which is the most
abundant plasma protein in teleost fishes [19]. Besides
being primarily involved in cholesterol metabolism in
mammals, ApoLP A-I has been shown to have a
regulatory role in the complement system by affecting
the assembly of the MAC in two different ways.
Firstly, specific and saturable binding sites for ApoLP
A-I and A-II are expressed by C9 polymers, and
their binding at this new site might interfere with
the assembly of C9 into the polyC9 tubule and
insertion into the cell membrane [20]. Secondly,
ApoLP A-I forms high density lipoprotein complexes
with clusterin, which then inhibits the C5b-9 assembly
by interfering with the binding of C5b67 to cell
membranes [21]. This form of clusterin may also be
involved in lipoprotein metabolism or lipid redis-
tribution [21,22]. Apolipoproteins A-I and A-II have
been shown to inhibit complement-mediated lysis of
human and sheep erythrocytes after C9 is bound to
membrane-associated C5b-8 complexes [23], and in
preliminary studies on cod, purified human ApoLP
A-I was found to significantly reduce the haemolytic
activity of cod sera [24].
Other related functions that have been attributed to
ApoLP involve the binding of LPS [25,26], antiviral
activity [27,28] and heparin binding activity implicated
in nerve regeneration processes [29]. ApoLP A-I has
neutralizing effects on the proinflammatory activity of
CRP and on CRP expression in normal plasma [30]. It
is also involved in regulating the cytokine network,
which needs to be tightly controlled by natural
inhibitory mechanisms due to potent activities in cell
growth and differentiation, development and repair
processes leading to the restoration of homeostasis
[31]. In cod plasma, ApoLP A-I appears to be
hydrophobically associated with cod C3, which might
explain its inhibition of haemolytic activity [4,24].
Experimental farming of Atlantic cod (Gadus
morhua L.) is being carried out in several countries,
and studies of the ontogenic development and activity
of non-specific and specific immune mechanisms are
highly relevant to evaluate the ability of cod larvae to
combat diseases. In ontogenic studies of the lymphoid
organs in cod, it was found that immunoglobulin-
producing cells were not present until about 58 days
after hatching [32] and thus, during the first 2–3
months, the cod is dependent on innate immune
parameters, including complement proteins, for the
defence against pathogens or opportunistic agents [33,
34]. The ontogeny of complement components has not
been extensively studied in fish, apart from recent
immunohistochemical studies on C3 in cod and
halibut ontogeny [7,8].
In the present study, the ontogenic mRNA
transcription of complement component C3 and
ApoLP A-I was monitored with in situ hybridisation
in a continuous sequence of cod eleutheroembryo and
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larvae, from 11 days postfertilisation until 57 days
posthatching, and compared with sections stained for
apoptotic cells with TUNEL. The findings are in
accordance with C3 detection at the protein level in
developing cod [7], confirming that C3 is indeed
expressed extrahepatically in cod; similarly, ApoLP
A-I is found to be transcribed widely.
2. Materials and methods
2.1. The fish and sampling
Cod (Gadus morhua L.) larvae were obtained from
the Marine Institute’s Experimental Fishfarm Staður,
Grindavık, Iceland. The rearing process of the larvae
has been described before [7].
Cod samples were taken at 11 days postfertilization
and at 4, 7, 14, 21, 28, 35, 43, 51 and 57 days
posthatching. Clusters of fertilized eggs and four
larvae for each age stage were collected. The samples
were fixed in 4% formalin in phosphate buffered
saline (PBS) and kept at 4 8C and embedded in
paraffin within 2 days. The paraffin embedded blocks
were stored at room temperature.
2.2. cDNA library and immunoscreening
A cDNA expression library was constructed in the
UNI-ZAP XR vector (Lambda ZAPw II vector, ZAP-
cDNAw Synthesis Kit, Catalog # 200400, Stratagene,
La Jolla, CA, USA), using cDNA synesized from cod
liver mRNA, which was isolated using Quick-Prepe
Total RNA Extraction Kit and mRNA Purification Kit
(Amersham Pharmacia Biotech, UK). To find genes
coding for cod C3, the cDNA library was immu-
noscreened with polyclonal mouse antibodies against
purified cod C3 [4]. The isolation of the cod ApoLP
A-I sequence has been described elsewhere [24].
After subsequent screening, immunopositive clones
were purified and converted into pBluescript phage-
mid particles using the ExAssiste helper phage and
SOLRe (non-suppressing Escherichia coli) strain in
accordance with the manufacturer’s instructions
(ZAP-cDNAwGigapackw III Gold Cloning Kit,
Stratagene, catalog.# 200450). Single colonies were
picked from agar plates and plasmids were isolated
with the QIAprep Spin Plasmid Miniprep Test Kit
(Qiagen, Valencia, CA, USA). A 3500 bp insert of
cod C3 was sequenced (ABI PRISMw Big Dyee
Terminator Cycle Sequencing Ready Reaction Kits,
PE Biosystems, CA, USA), using T3 and T7 plasmid
sequencing primers. The C3 sequence was aligned
with known C3 sequences using the ClustalW
programme [35] version 1–7 using default parameters.
2.3. In situ hybridisation
Internal primers were designed from the 3500 bp
cod C3 sequence to give a 550 bp PCR-product
(GenBank nr AY-739672), which was then cloned
into a pBluescript SK (C) transcription vector. The
ApoLP A-I containing vector (GenBank nr AY-
739673, [24]) was used without modification. Both
vectors were linearised enzymatically with NotI or
XhoI, followed by phenol extraction and precipitation
under RNAse free conditions. Run-off digoxigenine
(DIG) labelled sense- and anti-sense RNA transcripts
were transcribed using T3 and T7 RNA polymerase
according to the manufacturer’s instructions (DIG
RNA Labeling Kit, Roche, Germany).
In situ hybridisation was based on methods by
Komminoth [36] and Breitschopf and Suchanek [37]
and all solutions were treated with 0.1% Diethyl
pyrocarbonate (DEPC) and autoclaved before use. In
brief, paraffin embedded sections was dewaxed in
xylene and washed and rehydrated in sequences of
ethanol (100, 96 and 70%). The sections were
postfixed in 4% paraformaldehyde in TBS (50 mM
Tris pH 7.5, 150 mM NaCl) for 10 min at 4 8C,
washed in TBS and digested with 20 mg mlK1
proteinase K in TE buffer (50 mM Tris, 5 mM
EDTA, pH 8.0) for 30 min at 37 8C. After washing
in TBS for 3!10 min, the digestion was stopped by
incubating the sections in TBS at 4 8C for 5 min. The
sections were prehybridized in 4!SSC (600 mM
NaCl, 60 mM Sodiumcitrate, pH 7.0) and 50%
formamide for 15 min at 42 8C and thereafter
incubated with the linearized anti-sense probe
(sense-probe for negative control), which was kept
at 65 8C for 7 min before being added to the sample,
overnight in a humidity chamber at 40 8C (for C3
probes) or 42 8C (for ApoLP A-I probes), covered
with a DEPC-treated coverglass and wrapped in
parafilm. After incubation, any unbound probe was
washed off at 52 8C with 2!SSC in 50% formamide
Table 1
A schematic view for C3 and ApoLP A-I expression and TUNEL
staining in cod larvae development from 4 to 57 days postfertilisa-
tion
C3 d.p.h. ApoLP
A-I d.p.h.
TUNEL
d.p.h.
Liver 4–57 4–57 21–57
Skin 4–57 4–57 nd
Muscle 4–57 4–57 4–57
Braina 4–57 4–57 4–57
Eye 4–57 4–57 4–57
Chondrocytes 4–57 4–57
Spinal chord 4–57 4–57 21–57
Kidney 4–57 4–57
Intestines 4–57 4–57 21–57
Heart 4–57 14–57 nd
Pancreas 14–57 21–57 nd
Thymus 51–57 57–57
Apoptotic cells were not detected (nd) in skin, heart and pancreas in
these samples although the organs were present on the tissue
sections for TUNEL staining.a In neuronal tissue, a faint C3 expression was seen at 11 days
postfertilization.
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for 2!15 min, 1!SSC in 50% formamide for 2!15 min and at room temperature in 0.1!SSC for 2!30 min. The sections were then washed in TBS at
room temperature for 2!10 min and thereafter
blocked in 2% blocking reagent (Roche, 10% stock
solution prepared in 100 mM Malaic acid buffer,
150 mM NaCl2) in TBS for 30 min at room tempera-
ture followed by incubation for 2 h at room tempera-
ture with anti-DIG-AP Fab fragments (Roche) diluted
1/400 in 2% blocking reagent in TBS. Then the
sections were washed in TBS for 6!10 min and
colour detection was done with fast red (DAKO,
Denmark). The sections were background stained
with Mayer’s haematoxylin (Sigma, USA) and
mounted with Faramount (DAKO).
2.4. TUNEL staining for apoptotic cells
Paraffin embedded tissue sections of cod larvae
were stained with the DeadEnde Colorimetric
TUNEL system from Promega (USA). The system
detects apoptotic cells by measuring nuclear DNA
fragmentation. In brief, paraffin sections were
dewaxed, rehydrated in sequential ethanol washes
(100, 95, 85, 70 and 50% ethanol) and washed in
0.85% NaCl followed by PBS. The tissue was
postfixed in 4% paraformaldehyde in PBS at room
temperature for 15 min, digested with 20 mg mlK1
proteinase K for 15 min at room temperature and
refixed after washing with PBS by immersing in 4%
paraformaldehyde in PBS for 5 min at room tempera-
ture. The sections were washed and covered with
equilibration buffer from the kit and then incubated
with rTdT reaction mix for 1 h at 37 8C for end-
labelling. In the negative controls the rTdT enzyme
was omitted and replaced by dH2O. After end-
labelling the sections were washed in 2!SSC and
PBS, incubated with streptavidin HRP for 30 min and
colour development was done with DAB. Back-
groundstaining and mounting was as described before.
3. Results
3.1. Isolation of C3 positive clones
The 3500 bp cod C3 insert isolated from the cDNA
library, by immunoscreening with polyclonal mouse
anti-cod C3, started at position 588 corresponding to
human C3 and covered the N-terminal end of the C3
a-chain. When comparing the deduced amino acid
sequence to the N-terminal amino acid sequence
previously obtained for the a-chain of the purified cod
C3 [4], an exact match was found.
The subcloned 550 bp cod C3 insert, starting at
the COOH-end of the a-chain (GenBank nr.
AY739672), was compared to the corresponding
part of other known teleost C3 sequences, using
ClustalW. This part of the cod C3 showed a 49, 49, 48
and 45% identity to the corresponding part of C3 from
Japanese medaka (Oryzias latipes), rainbow trout
(Oncorhynchus myskiss), wolffish (Anarhichas minor)
and carp (Cyprinus carpio), respectively.
3.2. Transcription of C3 mRNA in cod larvae
The C3 mRNA transcription pattern was as follows
(summarized in Table 1 and pictured in Fig. 1).
A vague positive C3 mRNA signal was seen in
brain of cod embryo at 11 days postfertilization (not
shown).
On day 4 p.h. C3 mRNA was detected in the
hepatocytes of the liver (Fig. 1(a)), and in the neurons
of the spinal chord and surrounding muscle fibres
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(Fig. 1(b)). The corresponding negative controls are
shown in Fig. 1(a1) and (b1). A faint C3 mRNA signal
was also detected in the inner and outer ganglion layer
of the eye, and C3 mRNA was seen in the
chondrocytes and squamous epithelial cells of the
gut. A faint C3 mRNA signal was seen in the outer
myocardial layer of the atrium of the heart and in
tubule of kidney. C3 mRNA was also detected in
striated muscle fibres and in the skin (not shown).
On day 7 p.h. C3 mRNA was seen in neuronal
bodies of the brain, the spinal chord and in the
plexiform layer of the retina and inner and outer layer
of ganglion cells of the eye. C3 mRNA was also
detected in chondrocytes of cartilage, in columnar
epithelial cells in gut, striated muscle fibres and a low
transcription signal was seen in tubule of kidney and
the skin (not shown).
On day 14 p.h. C3 mRNA was seen in brain and
eye as before and in columnar epithelial cells of the
intestines. A weak transcription signal was seen in the
tubule and lymphomyeloid tissue of kidney and
hepatocytes of the liver, whereas the C3 mRNA
signal in small and large striated muscle fibres in the
tail and in chondrocytes of cartilage was strong. C3
mRNA was also detected in the outer myocardial
layer of the atrium of the heart and a strong signal was
seen in the skin (not shown).
On day 21 p.h. C3 mRNA was seen clearly in the
hepatocytes of the liver (not shown), in bodies of
neurons in the ganglion and plexiform layer of the
retina of the eye and in the photoreceptors of the eye
(Fig. 1(c)). A strong signal was seen in bodies of
neurons in the brain (Fig. 1(d)) and in chondrocytes in
cartilage in the head region (Fig. 1(e)). C3 mRNA was
also detected in the columnar epithelium of the gut
(Fig. 1(f)). The corresponding negative controls are
shown in Fig. 1(c1)–(f1). C3 mRNA was also seen in
exocrine cells of the pancreas (not shown).
On day 28 p.h. C3 mRNA was seen in the same
organs as before.
On day 35 p.h. C3 mRNA was detected in the
tubule and lymphomyeloid tissue of kidney (Fig. 1(g))
and a faint signal was also seen in the glomerulus (not
shown). At this stage a strong C3 mRNA signal was
seen in columnar epithelium in the intestines
(Fig. 1(h)) and the neurons in the spinal chord were
strongly positive (Fig. 1(i)) as well as bodies of
neurons in the brain (not shown). The islet of
Langerhans in pancreas showed a high level of C3
mRNA and lower signal was detected in the exocrine
cells of pancreas (Fig. 1(j)). The corresponding
negative controls are shown in Fig. 1(g1)–(j1).
On day 43 p.h. C3 mRNA was seen in bodies of
neurons in the brain, chondrocytes of cartilage,
columnar epithelium, liver hepatocytes, skeletal
muscle and spinal chord as before. At this stage C3
mRNA was also seen in the spleen (not shown).
On day 51 p.h. a strong C3 mRNA signal was
found in the brain, in chondrocytes in cartilage in fins,
in photoreceptors, inner and outer layer of ganglion
cells and neuronal bodies in the plexiform layer of the
retina of the eye, in hepatocytes of the liver and a
strong response was seen in the columnar epithelial
cells of the intestines and stomach (not shown).
On day 57 p.h. C3 mRNA was widely and evenly
distributed in the brain (Fig. 1(k)) and in the liver
hepatocytes (Fig. 1(l)). The corresponding negative
controls are shown in Fig. 1(k1) and (l1). Myofibrils in
the heart were positive and a strong signal was seen in
squamous epithelial cells in oesophagus, intestine and
stomach. C3 mRNA was also seen in chondrocytes
and striated muscle as well as in exocrine cells of the
pancreas. Neurons in the spinal chord were clearly
positive as well as neuronal bodies in the eye. In
kidney, a strong signal was seen in tubuli and
lymphomyeloid tissue and a faint signal in glomer-
ulus. C3 mRNA was for the first time in thymocytes of
the thymus (not shown).
3.3. Transcription of ApoLP A-I mRNA in cod larvae
The ApoLP A-I mRNA transcription pattern was
as follows (summarized in Table 1 and pictured in
Fig. 2).
On day 4 p.h. ApoLP A-I mRNA was seen in
neuronal bodies of the brain, the spinal chord and the
ganglion and plexiform layer of the retina of the eye.
ApoLP A-I mRNA also detected in tubule of kidney,
striated muscle fibres, chondrocytes of cartilage, skin,
hepatocytes of the liver and columnar epithelial cells
of the gut (not shown).
On day 7 p.h. ApoLP A-I mRNA was seen evenly
distributed in the brain (Fig. 2(a)) and in bodies of
neurons of the ganglion and plexiform layer of the
retina of the eye (Fig. 2(b)). The ApoLP A-I mRNA
signal was strong in the bodies of neurons of the spinal
Fig. 1. The detection of C3 mRNA in various organs of cod from 4 days posthatching until 57 days posthatching. Cells transcribing C3 mRNA
are stained with fast red and counterstain is with Mayer’s haematoxylin (blue). At 4 days posthatching: (a) hepatocytes in the liver; (b) neuronal
cells in the spinal chord and striated muscle cells; (2b) (a1) and (b1) are the corresponding negative controls. At 21 days posthatching:
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chord and was also seen in the surrounding striated
muscle (Fig. 2(c)). A low signal was seen in the liver
hepatocytes (Fig. 2(d)). The corresponding negative
controls are shown in Fig. 2(a1)–(d1). ApoLP A-I
mRNA was also seen in chondrocytes of cartilage and
in the skin (not shown).
On day 14 posthatching ApoLP A-I mRNA
transcription was clearly seen in striated muscle fibres
of the larvae tail (Fig. 2(e)), in chondrocytes in the
skull cartilage and in the neurons of the brain
(Fig. 2(f)). The corresponding negative controls are
shown in Fig. 2(e1) and (f1). A faint transcription
signal was seen in the tubuli of kidney and in
myofibrils of the heart as well as in exocrine cells of
the pancreas. The transcription signal was strong in the
skin and the signal in eye was as before (not shown).
On day 21 p.h. the ApoLP A-I mRNA transcription
signal was strong and evenly distributed in the
neuronal bodies of the brain. Transcription was seen
in the eye and chondrocytes as before and the signal
was strong in liver hepatocytes. ApoLP A-I mRNA
was also seen in the atrium and ventricle of the heart,
in exocrine cells of the pancreas, in squamous
epithelial cells of intestines and stomach, and in the
skin (not shown).
On day 28 p.h. ApoLP A-I mRNA was seen in the
brain, in photoreceptors and bodies of neurons in the
plexiform layer of the retina of the eye, in the liver
hepatocytes and in chondrocytes of cartilage.
On day 35 p.h. ApoLP A-I mRNA was seen, as
before, in the brain and spinal chord, chondrocytes of
cartilage, liver hepatocytes, striated muscle fibres,
intestine and in the kidney, both in tubule in
glomerulus. The exocrine cells of the pancreas
showed a low level of ApoLP A-I mRNA.
On day 43 p.h. ApoLP A-I mRNA was detected on
a low level in the tubuli and lymphomyoloid tissue of
kidney (Fig. 2(g)). A strong positive was seen in
chondrocytes in cartilage in the head region
(Fig. 2(h)). The corresponding negative controls are
shown in Fig. 2(g1) and (h1). ApoLP A-I mRNA was
also seen in the spleen (not shown).
(c) neuronal cells in the plexiform layer and the inner and outer layer of
chondrocytes in cartilage of the head; (f) columnar epithelial cells of the int
posthatching: (g) tubuli (pronephric tubuli) of kidney; (h) columnar epit
pancreas with C3 expressing cells in the islet of Langerhans in the pancre
posthatching: (k) neurons in the brain; (l) liver; (k1) and (l1) are the corre
3
On day 51 p.h. ApoLP A-I mRNA was detected in
neurons of spinal chord and surrounding muscle as
before (Fig. 2(i)) and in the plexiform layer and
photoreceptors in the eye (Fig. 2(j)). The correspond-
ing negative controls are shown in Fig. 2(i1) and (j1).
A strong positive was seen in the skin as well as in
neuronal bodies of the brain, chondrocytes and
striated muscle. ApoLP A-I mRNA was also seen in
thymocytes in thymus for the first time (not shown).
On day 57 p.h. ApoLP A-I mRNA signal was
strong in the columnar epithelium and the circular and
longitudinal muscles of the intestine (Fig. 2(k)). The
signal in liver hepatocytes reached a peak (Fig. 2(l)),
ApoLP A-I mRNA was clear in striated muscle fibres
(Fig. 2(m)) and the bodies of neurons in the brain
showed strong positive (Fig. 2(n)). The corresponding
negative controls are shown in Fig. 2(k1)–(n1).
ApoLP mRNA was abundant in tubuli and lympho-
myeloid tissue of kidney and was also seen in thymus
(not shown).
3.4. TUNEL staining
Cod larvae at 4, 7, 14, 21, 28, 35, 43, 51 and 57 d.p.h.
were TUNEL stained to detect cells undergoing
apoptosis (summarized in Table 1 and pictured in
Fig. 3). A few apoptotic cells were detected from 4
d.p.h. in the inner ganglion layer of the eye, in neuronal
bodies of the brain and in striated muscle fibres in the
tail. The number of apoptotic cells increased with
age and they were also found in liver, intestine, and
the spinal chord at 21 d.p.h. as well as in kidney,
chondrocytes and thymus at 57 days posthatching.
Fig. 3 displays apoptotic cells amongst neuronal bodies
of the brain at 28 d.p.h. (Fig. 3(a), (a1), see (a2) for
negative control), in striated muscle cells in the tail at 43
d.p.h. (Fig. 3(b), (b1), see (b2) for negative control), in
the ganglion layer of the eye at 51 d.p.h. (Fig. 3(c), (c1)
see (c2) for negative control) and in the ganglion layer
of the eye, neuronal bodies of the brain, chondrocytes of
cartilage, and in the intestine at 57 d.p.h. (Fig. 3(d)–(g),
see (d1)–(g1) for negative control).
ganglion cells in the retina of the eye; (d) neurons in the brain; (e)
estines. (c1)–(f1) are the corresponding negative controls. At 35 days
helial cells in the intestines; (i) nerve cells in the spinal chord; (j)
as. (g1) to (j1) are the corresponding negative controls. At 57 days
sponding negative controls.
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4. Discussion
The detection of mRNA for complement com-
ponent C3, the central protein of the complement
pathways, and the associated ApoLP A-I, a possible
control protein of the membrane attack complex, was
performed in parallel sections of cod embryo and
larvae using the in situ hybridisation technique.
Apoptosis was also determined in concurrent sections
using the TUNEL technique. The results are summar-
ized in Table 1.
C3 and ApoLP A-I were detected in various organs
from 11 days after fertilization until 57 days
posthatching (d.p.h.), which was in accordance with
former studies of protein expression, using immuno-
histological and Western blotting techniques [7,38].
In most organs (liver, muscle, brain, eye, spinal
chord, kidney and intestines) and in chondrocytes, C3
and ApoLP A-I were detected at the same develop-
mental stage. Exceptions were the C3, which was
detectable from 4 d.p.h. in the heart and the pancreas
while ApoLP A-I was not detected in these organs prior
to 14 and 21 d.p.h., respectively. These proteins were
commonly found in the same areas as apoptotic cells.
Apoptosis was seen in the brain, muscle and eye in early
development but later (O21 d.p.h.) in other organs.
The mRNA detection of C3 and ApoLP A-I was
seen in hepatocytes of cod liver in all larval stages
examined and increased with age. The liver
is generally considered to be the prime organ involved
in complement and ApoLP A-I synthesis. C3 mRNA
detection has been shown in the cytoplasm of the liver
hepatocytes of adult wolffish [39] and in mammals,
the liver is the main site of biosynthesis for the
majority of the fluid-phase complement components
[6]. Similarly, ApoLP A-I has been demonstrated
in the liver of fish, avian and mammalian species
[40–42].
Fig. 2. ApoLP A-I mRNA in various organs of cod from 7 days posthatchi
are stained with fast red and counterstain is with Mayer’s haematoxylin (b
inner and outer layer of ganglion cells and neurons in the plexiform layer i
hepatocytes of the liver. (a1) to (d1) are the corresponding negative control
cartilage and neurons in the brain. (e1) and (f1) are the corresponding negat
tissue in the kidney; (h) chondrocytes in cartilage, (g1) and (h1) are the co
bodies in the spinal chord; (j) neurons in the ganglion layer in the eye and p
At 57 days posthatching: (k) columnar epithelial cells and the circular an
skeletal muscle; (n) neurons in brain. (k1) to (n1) are the corresponding n
3
In addition to the liver detection of C3 and ApoLP
A-I, the present study clearly demonstrated an
extrahepatitic biosynthesis in all stages of cod larval
development. Lymphoid organs such as the kidney
and spleen contained C3 and ApoLP A-I mRNA from
(at least) 4 d.p.h. and the thymus showed positive
when present (at 51–57 d.p.h.). These messages were
also detected in other haematopoietic or lymphoid
organs of cod, like the heart and gut, throughout larval
development. The reason for the delayed appearance
of C3 in the pancreas and the later appearance of
ApoLP A-I in the heart and pancreas compared to C3
is not known.
Organs of the nervous system, the brain and spinal
chord, as well as the eye showed a strong transcription
of both mRNAs from early larval stages. A faint
positive for C3 was seen in the neuronal tissue of cod
embryo at 11 days after fertilization and C3 and
ApoLP A-I message were widely distributed in the
spinal chord and in nerve cells of all regions in the
brain from 4 d.p.h., and stayed at similar levels in all
the stages examined. Likewise, C3 and ApoLP A-I
were detected in muscle tissue, the striated inner and
outer muscle cells, and chondorcytes in the cartilage
of the head region and in the gills from 4 d.p.h.
onward. The results confirm the previous sign of
extrahepatitic synthesis of C3 protein in cod [7] and
bear out the close association of C3 and ApoLP A-I in
tissues as in plasma [4,24].
Although this is the first demonstration of an
extrahepatic synthesis of C3 and ApoLP A-I through-
out the development stages of a vertebrate species
such detection has been previously described in
isolated tissues of both adult and embryonic stages
of various animals. For example, C3 was detected in
amphibian limb and eye [43], in murine spleen [44]
and cartilage bone of rat [10] and local synthesis of
complement components in the mammalian brain has
ng until 57 days posthatching. Cells transcribing ApoLP A-I mRNA
lue). At 7 days posthatching: (a) Bodies of neurons in the brain; (b)
n the retina of the eye; (c) bodies of neurons in the spinal chord; (d)
s. At 14 days posthatching: (e) striated muscl; (f) chondrocytes in the
ive controls. At 43 days posthatching: (g) tubule and lymphomyoloid
rresponding negative controls. At 51 days posthatching: (i) neuronal
hotoreceptors. (i1) and (j1) are the corresponding negative controls.
d longitudinal muscles of the intestine; (l) hepatocytes of liver; (m)
egative controls.
Fig. 3. TUNEL staining of cod larvae sections at 28, 43, 51 and 57 days posthatching, apoptotic cells are stained brown/black and counterstain is with
Mayer’s haematoxylin (blue). At 28 days posthatching: (a) neuronal cells in the brain with some apoptotic cells, (a1) shows some apoptotic neuronal
cells in magnification, (a2) is the negative control. At 43 days posthatching: (b) striated muscle with some apoptotic cells, (b1) is a magnification of
apoptotic cells and (b2) is the negative control. At 51 days posthatching: (c) neuronal cells in the ganglion layer in the retina of the eye with some
apoptotic cells, (c1) is a magnification of apoptotic neuronal cells and (c2) is the negative control. At 57 days posthatching: (d) cod eye lens with some
apoptotic cells; (e) cod brain with some apoptotic neuronal cells; (f) cod cartilage in the head region with some apoptotic chondrocyte cells; (g) cod
liver with some apoptotic hepatocytes; (h) cod intestine with apoptotic cells. (d1) to (h1) are the corresponding negative controls.
S. Lange et al. / Developmental and Comparative Immunology xx (2005) 1–1310
DTD 5 ARTICLE IN PRESS
been demonstrated [6,45,46]. ApoLP A-I has been
detected in kidney, brain and spleen of chicken [40],
in foetal human kidney, pancreas, stomach and
gonads [43] and in salmon muscle [41].
The present study is in accordance with the
suggestion that as well as being an important immune
mechanism, the complement system participates in
organogenesis and homeostasis of the developing
S. Lange et al. / Developmental and Comparative Immunology xx (2005) 1–13 11
DTD 5 ARTICLE IN PRESS
embryo. Also in adult vertebrates and urodelians,
complement components can be involved in metamor-
phosis, regeneration of injured organs and maintaining
tissue homeostasis [10,43,47]. In the developing
embryo apoptosis and phagocytosis have an important
role in organ formation and in the establishment of the
nervous and immune system [18]. Cells showing
restricted phagocytic activity have been demonstrated
in cod larvae at 4 d.p.h., showing full activity at 14
d.p.h. [48]. In the present study, using the TUNEL
technique, apoptosis was seen in several organs during
larval development and close to cells showing positive
for mRNA of C3 and ApoLP A-I. This was seen in the
early stages, from 4 d.p.h., in muscle tissue, brain and
eye and in later stages in other organs such as the spinal
chord, intestines, liver and the thymus (Table 1).
TUNEL staining visualizes cells that are in late
apoptosis but does not detect early stages of cell
death or cells that do not have the nuclei in the plane of
section, which in some instances could account for the
weak staining [18].
Few other studies have been made of apoptosis
during the ontogeny of fish. In sea bass, it has been
shown that apoptosis takes place during the develop-
ment of the thymus. The apoptotic cells were less
numerous in juvenile specimens than in older speci-
mens but the pattern distribution was the same [49].
The involvement of apoptosis in the development or
metamorphosis of other species has, however,
received some attention and these studies bear out
the present findings [16,18,50–53].
The detection of C3 and ApoLP A-I in close
proximity to apoptotic cells in the present study might
suggest that these phenomena play a part in the
ontogeny of cod. Such a co-operation between the
complement system and programmed cell death has,
for example, been found in studies of murine
embryonic cells [16]. It has been suggested that the
local production of components of the complement
system plays a role in the opsonization of apoptotic
cells during the late phase of apoptosis at sites where
the local rate of apoptosis is high and the phagocytic
capability relatively low or impaired [17,54,55].
ApoLP A-I is hydrophobically associated with C3
in cod plasma and there are indications that this
association blocks the lytic pathway [4,20,24]. In
human plasma, ApoLP A-I has been isolated in
conjunction with clusterin [21]. Clusterin shows an
affinity for cell membranes, especially of damaged,
abnormal or dying cells and it has been suggested that
clusterin and ApoLP A-I clusterin complexes could
assist in the uptake of membrane lipids originating
from apoptotic cells [21]. It has also been hypothesed
that these complexes could have a protective role by
inhibiting the complement-mediated cytolysis and
maintaining minimal inflammation during apoptosis
[22]. It can be speculated that ApoLP A-I in
conjunction with C3, might have a similar protective
function in the development of cod.
In conclusion, the early and wide spread occurance
of complement component C3 and the possibly
regulating protein ApoLP A-I in association with
apoptosis suggest that the cooperation of these factors
may play an important role in the organogenesis and
homeostasis during the larval development of cod.
The early expression of C3 and ApoLP A-I in immune
defence is probably also of considerable importance
in view of the extended period (O60 d.p.h.) until the
cod fry has attained full immunological competence
[32]. These results could be of value when considering
prophylactic measures in cod larval aquaculture and
will hopefully contribute to the understanding of C3
and ApoLP A-I functions during development.
Acknowledgements
The authors wish to thank Agnar Steinarsson,
Matthıas Oddgeirsson and the staff at Stadur,
Grindavık, Iceland, for providing the fish and
sampling facilities. Thanks are also due to Margret
Jonsdottir at Keldur, Institute for Experimental
Pathology University of Iceland, for preparation of
cod larvae samples and tissue sections. Thanks to
Professor Jurg A. Schifferli, University Hospital
Basel, Switzerland, for providing research facilities
for part of this work, and to Brigitte Schneider
and Kwok Min Hui, University Hospital Basel, for
technical assistance. This work was supported by the
EC grant FISHAID QLRT-1999-31076, The Icelandic
Ministry for Fisheries, The Icelandic Research
Council, The European Moleacular Biology Organis-
ation (EMBO), The Nordic Organisation for Fish
Immunology (NOFFI), The International Union of
Biochemistry and Molecular Biology (IUBMB) and
the Swiss National Science Foundation.
S. Lange et al. / Developmental and Comparative Immunology xx (2005) 1–1312
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