First published online February 13, 2009
Journal of Experimental Biology 212, 627-638 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026286
Diverse cell-specific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish
Andrew R. Cossins1,*,
Daryl R. Williams1,
Nick S. Foulkes2,
Michael Berenbrink1 and
Anja Kipar3
1 School of Biological Sciences, University of Liverpool, Crown Street,
Liverpool L69 7ZB, UK
3 Department of Veterinary Pathology, University of Liverpool, Crown Street,
Liverpool L69 7ZB, UK
2 The Research Centre, Karlsruhe, Germany
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Fig. 1. (A) Representative western analysis of tissue Mb proteins. Gill and liver
display an identical molecular mass, which, based on the sequenced cDNA
(Fraser et al., 2006 ), has a
predicted molecular mass of 15.744 kDa. Brain displays a larger protein,
consistent with a predicted molecular mass of 16.174 kDa, together with a band
at lower molecular mass that migrates alongside the single band (cMb1)
observed in other tissues. The higher abundance of cMb2 compared with cMb1
protein in brain correlates closely to estimated transcript levels that have
been reported previously (Fraser et al.,
2006). Note that the presence of the less-abundant cMb1 isoform in
brain is only visible in brain samples abundant in myoglobin. Protein loading
was not equivalent between tissues. (B) Quantitative comparison of Mb protein
expression in tissues of the common carp determined using immuno-detection on
a dot-blot. The vertical axis represents arbitrary densitometric values. The
heart supernatant was diluted 100x in order to provide for band
densities that were similar to the undiluted supernatants of the other
tissues. The horizontal dashed lines thus represent 1 and 0.5% of the mean
heart Mb levels (N=4, where each sample was taken from a different
specimen). (C) The effects of chronic (5 day) hypoxia treatment upon the
tissue Mb levels relative to those in normoxia controls. Samples were
subjected to western analysis, and each immunoblot contained normoxia- and
hypoxia-treated specimens for one tissue, together with a 100x diluted
heart extract as a positive control. Bands were analysed densitometrically and
values were expressed relative to the mean for the normoxia control for each
tissue (dashed line, N=5, except liver where N=4;
*P<0.01 and **P<0.001).
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Fig. 2. Carp myoglobin protein and mRNA expression in heart (A,B) and epaxial
muscle (C,D). (A) All cardiac myocytes exhibit strong, diffuse cMb protein
expression. In addition, endothelial cells in small capillaries stain positive
for cMb (arrows). (B) cMb mRNA expression is seen in all myocytes,
but often as a focal cytoplasmic signal (arrows). (C) In the epaxial skeletal
muscle, a small proportion of myofibres exhibit diffuse cytoplasmic protein
expression (black arrows). White arrow: myoseptum. Inset: a cross-section of
muscle shows the high variation in staining intensity between myofibres.
(D)cMb mRNA expression is seen as a focal cytoplasmic signal, mainly
at the periphery of the fibre (black arrows), close to the myoseptum (white
arrow). In A and C, rabbit anti-cMb peptide was used; in B and D, RNA-ISH with
the cMb1/2 riboprobe was used. Scale bars indicate 10 µm for A and
B, and 20 µm for C and D.
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Fig. 3. Myoglobin protein and mRNA expression in liver and pancreas of carp. (A)
cMb protein expression is most intense in endothelial cells lining the
sinusoids (large arrows) but is also seen in endothelial cells of some larger
arteries (large arrow labelled A). Hepatocytes generally exhibit faint-to-weak
cytoplasmic and occasional nuclear staining (small arrows). In particular, in
hypoxic carp, multifocal areas of hepatocytes that exhibit strong cMb staining
are apparent (inset). The exocrine pancreatic epithelial cells (P) generally
show faint cytoplasmic cMb expression (arrowheads). (B)cMb mRNA
expression is variable in hepatocytes, represented by an apparent lack of
signal up to a strong cytoplasmic signal (left inset). Occasional endothelial
cells, in particular of central veins, exhibit a signal (right inset, arrow).
The exocrine pancreatic epithelial cells (P) generally show moderate
cytoplasmic mRNA expression (arrows). In A, rabbit anti-cMb peptide was used;
in B, RNA-ISH with the cMb1/2 riboprobe was used. Scale bars indicate
20 µm.
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Fig. 4. Myoglobin protein (A,C) and mRNA (B,D: cMb1/2, E: cMb2)
expression in the carp brain. (A,B) Cerebellum. (A) cMb protein expression is
most intense in endothelial cells of capillaries (large arrows) and small
blood vessels (inset). Weak-to-moderate cytoplasmic staining is seen in some
Purkinje cells (small arrow) and in neuronal cell processes (arrowheads).
Erythrocytes are negative for cMb (inset). (B)cMb1/2 mRNA expression
is seen in occasional neurons in the molecular layer (arrowhead), in some
Purkinje cells (arrows) and in all cells in the granular layer (white arrow).
(C,D) Brain stem. (C) cMb protein expression is seen in vascular endothelial
cells (large arrow), in the cytoplasm of large motor neurones (arrowheads) and
in cell processes (small arrows). (D) Large motor neurones (arrows) and
vascular endothelial cells (arrowheads) are also positive for cMb1/2
mRNA. (E) Cortex. cMb2 mRNA expression is very limited and restricted
to small aggregates of neurons (arrows). In A and C, rabbit anti-cMb peptide
was used; in B and D, RNA-ISH with the cMb1/2 riboprobe was used; in
E, RNA-ISH with the cMb2 riboprobe was used. Scale bars indicate 20
µm for A, B and E; those in the inset to A, and in C and D are 10
µm.
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Fig. 5. Myoglobin protein (A) and mRNA (B) expression in the carp gill. (A) On the
left, an overview shows cMb protein expression in the lamellae (L), whereas
the cartilage (C) is negative (scale=20 µm). On the right, a magnified view
of the secondary lamellae highlights intense diffuse cytoplasmic cMb protein
expression by pillar cells (white arrows) and lamellar epithelial cells (black
arrows), whereas staining of chloride cells is generally less intense
(arrowheads; scale=10 µm). (B) On the left, an overview shows cMb
mRNA expression in the lamellae (L) and the chondrocytes in the
cartilaginous (C) skeleton (scale=20 µm). On the right, a closer view of
the secondary lamellae highlights intense diffuse cytoplasmic cMb
mRNA expression by lamellar epithelial cells (black arrows) and chloride cells
(arrowheads), whereas the signals in pillar cells are predominantly in the
flanges (red arrow; scale=10 µm). In A rabbit anti-cMb peptide was used; in
B, RNA-ISH with the cMb1/2 riboprobe was used.
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Fig. 6. Myoglobin protein (A,B) and mRNA (C,D) expression in the carp kidney. (A,B)
Mb protein expression is seen in tubular epithelial cells and was represented
by a diffuse cytoplasmic and often nuclear staining of individual (arrowheads)
or all epithelial cells (arrow) in a tubular cross-section. Glomerula (G) were
negative for Mb. (B) An area of haematopoietic tissue is included where
several haematopoietic cells expressed Mb protein (arrowheads). Capillary
endothelial cells expressing Mb protein were also observed (arrow). T, tubule;
G, glomerulum. (C,D) RNA-ISH showed a pattern of cMb mRNA expression
similar to the pattern of protein expression. In addition to tubular
epithelial cells (arrows), haematopoietic cells (arrowheads) exhibited a
diffuse cytoplasmic signal. In A and B, rabbit anti-Mb peptide was used; in C
and D, RNA-ISH with the cMb1/2 riboprobe was used. For A and C scale
bars indicate 20 µm and for B and D they indicate 10 µm.
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Fig. 7. Myoglobin protein (A,B) and mRNA (C,D) expression in the carp intestine.
(A) Intense cMb protein expression is seen in endothelial cells of capillaries
in all layers of the intestinal wall (rugae and smooth muscle layers, arrows).
(B) Higher magnification of submucosa and muscle layers (M) confirms
endothelial cMb protein expression (arrows) and weak staining for cMb in
smooth muscle fibres. (C) Endothelial cells of capillaries in all layers of
the intestinal wall exhibited cMb mRNA (black arrows). In addition,
neurones in the myenteric plexus (white arrows) and intestinal epithelial
cells (arrowhead) stain positive. (D) Higher magnifications of the submucosa
and smooth muscle layers confirmed cMb mRNA expression by neurones in
submucosal (arrow on left picture) and intermyenteric (arrows on right
picture) plexi and capillary endothelial cells (arrowheads on left picture).
In A and B, rabbit anti-cMb peptide was used; in C and D, RNA-ISH with the
cMb1/2 riboprobe was used. Scale bars indicate 40 µm for A, 20
µm for B, 40 µm for C and 10 µm for D.
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Fig. 8. Myoglobin protein and mRNA expression in various tissues of zebrafish. (A)
All cardiac myocytes exhibit strong Mb protein expression. (B) In the epaxial
skeletal muscle, myofibres (M) exhibit diffuse cytoplasmic Mb protein
expression. Capillary endothelial cells stain strongly positive (arrows). (C)
In the liver, strong cMb protein expression is seen in endothelial cells
lining the sinusoids (arrows). Hepatocytes stain negative. (D) The gills show
cMb protein expression by pillar cells (arrow) and lamellar epithelial cells
(arrowhead). (E) Brain cortex. cMb protein is seen in neuronal cell bodies
(large arrow) and cell processes (small arrows) as well as in capillary
endothelial cells (arrowheads). (F) RNA-ISH identifies cMb mRNA
expression in several types of neurones, such as Purkinje cells in the
granular layer in the cerebellum (arrows), large motor neurones in the
hypothalamus (inset) and in capillary endothelial cells (arrowhead). In
A–E, the PAP method and rabbit anti-cMb peptide were used; in F, RNA-ISH
with the cMb1/2 riboprobe was used. Scale bars indicate 20 µm for
A and F, 10 µm for B–E.
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