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DTD 5 ARTICLE IN PRESS

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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S. Lange et al. / Developmental and Comparative Immunology xx (2005) 1–132


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

S. Lange et al. / Developmental and Comparative Immunology xx (2005) 1–13 3


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.



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



(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:





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.



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.



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



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.



References

[1] Sunyer JO, Lambris JD. Evolution and diversity of the

complement system of poikilothermic vertebrates. Immunol

Rev 1998;166:39–57.

[2] Cole DS, Morgan BP. Beyond lysis: how complement

influences cell fate. Clin Sci 2003;104:455–66.

[3] Del Rio-Tsonis K, Tsonis PA, Zarkadis IK, Tsagas AG,

Lambris JD. Expression of the third component of comp-

lement, C3, in regenerating limb blastema cells of urodeles.

J Immunol 1998;161:6819–24.

[4] Lange S, Dodds AW, Magnadottir B. Isolation and character-

isation of complement component C3 from Atlantic cod

(Gadus morhua L.) and Atlantic halibut (Hippoglossus

hippoglossus L.). Fish Shellfish Immunol 2004;16(2):227–39.

[5] Law SKA, Dodds AW. The internal thioester and the covalent

binding properties of the complement proteins C3 and C4.

Protein Sci 1997;6:263–74.

[6] Morgan BP, Gasque P. Expression of complement in the brain:

role in health and disease. Immunol Today 1996;17:461–6.

[7] Lange S, Bambir S, Dodds AW, Magnadottir B. The ontogeny

of complement component C3 in Atlantic Cod (Gadus morhua

L.)—an immunohistochemical study. Fish Shellfish Immunol

2004;16:359–67.

[8] Lange S, Bambir S, Dodds AW, Magnadottir B. An

immunohistochemical study of complement component C3

in juvenile Atlantic halibut (Hippoglossus hippoglossus L.).

Dev Comp Immunol 2004;28:593–601.

[9] Colten HR. Immunology. Drawing a double-edged sword.

Nature 1994;371:474–5.

[10] Andrades JA, Nimini ME, Becerra J, Eisenstein R, Davis M,

Sorgente N. Complement proteins are present in developing

endochondral bone and may mediate cartilage cell death and

vascularization. Exp Cell Res 1996;227:208–13.

[11] Elward K, Gasque P. ‘Eat me’ and ‘Don’t eat me’ signals

govern the innate immune response and tissue repair in the

CNS: emphasis on the critical role of the complement system.

Mol Immunol 2003;40:85–94.

[12] Rooney IA, Oglesby TJ, Atkinson JP. Complement in human

reproduction—activation and control. Immunol Res 1993;12:

276–94.

[13] Mastellos D, Lambris JD. Complement: more than a guard

against invading pathogens? Trends Immunol 2002;23(10):

485–91.

[14] Ratajczak J, Reca R, Kucia M, Majka M, Allendorf DJ,

Baran JT, et al. Mobilization studies in mice deficient in either

C3 or C3a receptor (C3aR) reveal a novel role for complement

in retention of hematopoietic stem/progenitor cells in bone

marrow. Blood 2004;103(6):2071–8.

[15] Tsuji S, Kaji K, Nagasawa S. Activation of the alternative

pathway of human complement by apoptotic human umbilical

vein endothelial cells. J Biochem 1994;116:794–800.

[16] Kircheis R, Kircheis L, Oslhima H, Kohchi C, Soma G,

Mizuno D. Selective lysis of early embryonic cells by the

alternative pathway of complement—a possible mechanism

for programmed cell death in embryogenesis. In Vivo 1996;

10(4):389–403.

[17] Nauta AJ, Daha MR, van Kooten C, Roos A. Recognition and

clearance of apoptotic cells: a role for complement and

pentraxins. Trends Immunol 2003;24(3):148–54.

[18] Zakari Z, Lockshin RA. Cell death during development.

J Immunol Methods 2002;265:3–20.

[19] Amthauer R, Villanueva J, Vera M, Concha M, Krauskopf M.

Characterisation of the major plasma apolipoproteins of the

high density lipoprotein in the carp (Cyprinus carpio). Comp

Biochem Physiol B 1989;92:787–93.

[20] Hamilton KH, Zhao J, Sims PJ. Interaction between

apolipoproteins A-I and A-II and the membrane attack

complex of complement. Affinity of the apolipoproteins for

polymeric C9. J Biol Chem 1993;268:3632–8.

[21] Jenne DE, Lowin B, Peitsch MC, Bottcher A, Schmitz G,

Tschopp J. Clusterin (Complement lysis inhibitor) forms a

high density lipoprotein complex with apolipoprotein A-I in

human plasma. J Biol Chem 1991;266:11030–6.

[22] French LE, Wohlwend A, Sappino A-P, Tschopp J,

Schifferli JA. Human clusterin gene expression is confined

to surviving cells during in vitro programmed cell death. J Clin

Invest 1994;93:877–84.

[23] Packman CH, Rosenfeld SK, Leddy JP. High-density

lipoprotein and its apolipoproteins inhibit cytolytic activity

of compmplement. Studies on the nature of inhibitory moiety.

Biochim Biophys Acta 1985;812(1):107–15.

[24] Magnadottir B, Lange S. Is Apolipoprotein A-I a regulating

protein for the complement system of cod (Gadus morhua L.)?

Fish Shellfish Immunol 2003;16:265–9.

[25] Flegel WA, Baumstark MW, Weinstock C, Berg A,

Northoff H. Prevention of endotoxin-induced monokine

release by human low- and high-density lipoproteins and by

apolipoprotein A-I. Infect Immun 1993;61:5140–6.

[26] Juan MA, Xue-Ling LIAO, Bin LOU, Man-Ping WU. Role of

Apolipoprotein A-I in protecting against endotoxin toxicity.

Acta Biochimica et Biophysica Sinica 2004;36(6):419–24.

[27] Shrinivas RV, Birkendal B, Owens RJ, Anantharamaiah GM,

Sergrest JP, Compans RW. Antiviral effects of apolipoprotein

A-I and its synthetic amphipathic peptide analogs. Virology

1990;176:48–57.

[28] Singh IP, Chopra AK, Copperhaver DH,

Ananatharamaiah GM, Baron S. Lipoproteins account for

part of the broad non-specific anti-viral activity of human

serum. Antiviral Res 1999;42:211–8.

[29] Harel A, Fainaru M, Rubinstein M, Tal N, Swartz M. Fish

apolipoprotein A-I has heparin binding activity: implication

for nerve regeneration. J Neurochem 1990;55(4):1237–43.

[30] Wadham C, Albanese N, Roberts J, Wang L, Bagley CJ,

Gamble JR, et al. High-density lipoproteins neutralize C-

reactive protein proinflammatory activity. Circulation 2003;

109(17):2116–22.

[31] Burger D, Dayer J-M. Cytokines, acute-phase proteins, and

hormones. IL-1 and TNF-a production in contact-mediated

activation of monocytes by T lymphocytes. Ann NY Acad Sci

2002;966:464–73.

[32] Schrøder MB, Villena AJ, Jørgensen TO. Ontogeny of

lymphoid organs and immunoglobulin producing cells in



Atlantic cod (Gadus morhua L.). Dev Comp Immunol 1998;

22:507–17.

[33] Alexander JB, Ingram GA. Noncellular nonspecific defense

mechanisms of fish. Annu Rev Fish Dis 1992;2:249–79.

[34] Ellis AE. Innate host defence mechanisms of fish against

viruses and bacteria. Dev Comp Immunol 2001;25:827–39.

[35] Thompson J, Higgins D, Gibson T. Clustal W: improving the

sensitivity of progressive multiple sequence alignment

through sequence weithting, position-specific gap penalties

and weight matrix choice. Nucl Acid Res 1994;22:4673–80.

[36] Komminoth P. Detection of mRNA in tissue sections using

DIG-labelled RNA and oligonucleotide probes. In: Grune-

wald-Janho S, Keesey J, Leous M, van Miltenburg R,

Schroeder C, editors. Nonradioactive in situ hybridization

application manual. 2nd ed. Germany: Boehringer Mannheim

GmbH, Biochemica; 1996.

[37] Breitschopf H, Suchanek G. Detection of mRNa on paraffin

embedded material of the central nervous system with DIG-

labelled RNA probes. In: Grunewald-Janho S, Keesey J,

Leous M, van Miltenburg R, Schroeder C, editors. Non-

radioactive in situ hybridization application manual. 2nd ed.

Germany: Boehringer Mannheim GmbH, Biochemica; 1996.

[38] Magnadottir B, Lange S, Steinarsson A, Guðmundsdottir G.

The ontogenic develpment of cod (Gadus morhua L.) analysed

by SDS PAGE and Western blotting. Comp Biochem Physiol

B 2004;139:217–24.

[39] Abelseth TK, Stensvag K, Espelid S, Nygaard R, Ellingsen T,

Bogwald J, et al. The spotted wolfish (Anarhichas minor

Olafsen) complement component C3: isolation, characteriz-

ation and tissue distribution. Fish Shellfish Immunol 2003;

15(1):13–27.

[40] Byrnes L, Luo CC, Li WH, Yang CY, Chan L. Chicken

apolipoprotein A-I: cDNA sequence, tissue expression and

evolution. Biochem Biophys Res Commun 1987;128(1):

485–92.

[41] Powell R, Higgins DG, Wolff J, Byrnes L, Stack M, Shar PM,

Gannon F. The salmon gene encoding apolipoprotein A-I:

cDNA sequence. Tissue expression and evolution. Gene 1991;

104(2):155–61.

[42] Zannis VI, Cole FS, Jackson CL, Kurnit DM,

Karathanasis SK. Distribution of apolipoprotein A-I, C-II, C-

III and E mRNA in fetal human tissues. Timedependent

induction of apolipoprotein E mRNA by cultures of human

monocyte-macrophages. Biochemistry 1985;24(16):4450–5.

[43] Kimura Y, Madhavan M, Call MK, Santiago W, Tsonis PA,

Lambris JD, et al. Expression of complement 3 and

complement 5 in newt limb and lens regeneration.

J Immunol 2003;170(5):2331–9.

[44] Fischer MB, Ma M, Nelson C, Hsu N, Carroll MC. Local

synthesis of C3 within the splenic lymphoid compartment can

reconstitute the impaired immune response in C3-deficient

mice. J Immunol 1998;160(16):2619–25.

[45] Barnum SR. Complement biosynthesis in the central nervous

system. Crit Rev Oral Biol Med 1995;6:132–46.

[46] Gasque P, Dean YD, McGreal EP, VanBeek J, Morgan BP.

Complement components of the innate immune system in

health and disease in the CNS. Immunopharmacology 2000;

49:171–86.

[47] Mastellos D, Papadimitriou JC, Franchini S, Tsonis PA,

Lambris JD. A novel role of complement: mice deficient in the

fifth component of complement (C5) exhibit impaired liver

regeneration. J Immunol 2001;166:2479–86.

[[48] Gudmundsdottir S, Gudmundsottir BK, Bjornsdottir B,

Magnadottir B. Increased suvival of cod larvae bathed with

LPS or a bacterium of Vibrio sp. Sixth International

symposium on fish immunology, Turku, Finland 2004 p. 34.

[49] Albelli L, Baldassini MR, Mschini R, Mastrolia L. Apoptosis

of thymocytes in developing sea bass Dicentrarchus labrax

(L.). Fish Shellfish Immunol 1998;8:13–24.

[50] Kerr JFR, Harmon BV, Searle JW. An electron microscope

study of cell deletion in the anuran tadpole tail during

spontaneous metamorphosis with special reference to apopto-

sis of striated muscle fibres. J Cell Sci 1974;14:571–85.

[51] Appleby DW, Modak SP. DNA degradation in terminally

differentiating lens fiber cells from chick embryos. Proc Natl

Acad Sci USA 1977;74:5579–83.

[52] Hengartner MO, Horvitz HR. Programmed cell death in

Caenorhabditis elegans. Curr Opin Genet Dev 1994;4:581–6.

[53] Nijhawan D, Honarpour N, Wang X. Apoptosis in neural

develpment and disease. Annu Rev Neurosci 2000;23:73–87.

[54] Ezekowitz RA, Sim RB, Hill M, Gordon S. Local opsonization

by secreted macro-phage complement components. Role of

receptors for complement in uptake of zymosan. J Exp Med

1984;159:244–60.

[55] Gaipl US, Kuenkele S, Voll RE, Beyer TD, Kolowos W,

Heyder P, et al. Complement binding is an early feature of

necrotic and a rather late event during apoptotic cell death.

Cell Death Differ 2001;8:327–34.

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