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Pediatric Endocrinology and The Ilyssa Center for Molecular and Cellular Endocrinology (B.D.R., M.A.L.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; and Department of Zoology (G.M.W., C.V.S.), North Carolina State University, Raleigh, North Carolina 27695
Address all correspondence and requests for reprints to: Dr. Buel D. Rodgers, Pediatric Endocrinology, The Johns Hopkins University School of Medicine, 600 North Wolf Street, Park 211, Baltimore, Maryland 21287. E-mail: drodgers{at}jhmi.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In mammalian skeletal muscle, a recently identified member of the transforming growth factor-ß (TGFß) superfamily, appropriately named myostatin (MSTN), is believed to repress skeletal muscle growth by inhibiting both muscle cell hypertrophy and hyperplasia (7). Individual muscle fibers of homozygous MSTN knockout mice are double or triple in mass compared with comparable fibers isolated from their heterozygous and wild-type littermates (9). This "double muscling" phenotype is also characteristic of two domestic breeds of cattle, the Belgian Blue and the Piedmontese, which possess mutant alleles for MSTN (10, 11, 12, 13). Although MSTN null mice weighed approximately 30% more than the wild-type mice, total body fat content was equal in both groups. Thus, the growth inhibitory effects of MSTN in domesticated mice, and possibly in many mammals, appear to be limited primarily to skeletal muscle, as its expression was not detected in other fiber types or in other organs.
To date, MSTN complementary DNA (cDNA) clones have been isolated from seven different mammalian and two avian species as well as from zebra fish (13). However, the characterization of additional orthologs from nonmammalian models and in particular from commercially important species is unfortunately lacking, especially considering the potential contributions of such studies to the fields of comparative biology and aquaculture. If the atrophic actions of mammalian MSTN are determined to be conserved in fish, the successful manipulation of MSTN expression and/or its bioactivity in commercially important fishes will undoubtedly impact the economic, biotechnological, and political communities that rely on aquaculture. Reported herein is the isolation and characterization of MSTN cDNA clones from the skeletal muscle of two commercially important fish: the tilapia Oreochromis mossambicus and the white bass Morone chrysops.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of MSTN cDNA clones
Total RNA was isolated from tilapia epaxial skeletal
muscle with TRIzol reagent, and first strand cDNA was generated with
Superscript reverse transcriptase and oligo(deoxythymidine) primers
(all from Life Technologies, Inc.). Degenerate primers
[forward, 5'-GAT C(C/T)C TGA AA(A/C) T(C/T)G AC(G/A) TGA AC(G/C) CAG
G-3'; reverse, 5'-TGA GCA (C/G)CC (G/A)CA ICG (A/G)TC TAC TAC C-3']
derived from a compilation of all known MSTN cDNA sequences were used
to amplify a 500-bp partial clone via PCR that corresponds to the
latter half of the open reading frame. After an initial denaturing
period of 4 min at 94 C, cDNA was amplified for 30 cycles of 94 C for
30 sec, 52 C for 30 sec, and 72 C for 1 min. A final extension period
for 5 min at 72 C was also included. The 500-bp PCR product was then
gel purified, subcloned into the pcDNA3.1-TOPO TA cloning vector
(Invitrogen, San Diego, CA), and sequenced. The remaining
portions of the open reading frame and part of the 5'-untranslated
region were obtained with the rapid amplification of 5'-cDNA ends
system (Life Technologies, Inc., Gaithersburg, MD) using
the reverse primer listed above as well as the complement to the
forward degenerate primer. Full-length MSTN coding sequences for both
tilapia and white bass were then isolated by RT-PCR with gene-specific
primers derived from the partial tilapia (t) sequences (tMSTN forward,
5'-CCA CAC TTC ACA CCT AAG AGA CAA TGC-3'; tMSTN reverse, 5'-TGA GCA
GCC GCA GCG GTC TAC TAC C-3'). Skeletal muscle cDNA from both species
was amplified as described above, but with an annealing temperature of
55 C. The approximately 1.1-kb amplicons were then subcloned into the
pcDNA3.1/V5/His/TOPO TA cloning vector (Invitrogen) and
sequenced.
Southern, Northern, and Western blotting
Tilapia genomic DNA was isolated from whole blood that was
initially frozen, thawed, and then centrifuged at 3500 x
g for 15 min at 4 C. The pelleted debris was resuspended in
10 mM Tris (pH 8.0), 10 mM
EDTA, and 300 mM NaCl/0.2% SDS and incubated for
16 h at 37 C with 100 µg/ml proteinase K. Contaminating protein
was then precipitated with saturated NaCl, and genomic DNA was isolated
by three consecutive extractions with phenol-chloroform-isoamyl alcohol
(50:49:1). DNA (10 or 20 µg) was digested separately with
BamHI and EcoRI for 24 h. Digested DNA was
electrophoresed on a 1.0% agarose gel, transferred to a charged nylon
membrane, and hybridized to a radiolabeled tMSTN cDNA probe for 16
h at 42 C in a buffer containing 6 x SSC (standard saline
citrate), 50% formamide, and 5 x Denhardt’s solution.
Polyadenylated RNA was isolated with the PolyATract mRNA Isolation
System (Promega Corp., Madison, WI) from skeletal muscle
total RNA, and 5 µg were electrophoresed on a 1.2% agarose and
formaldehyde gel, then transferred and probed as described. Both
membranes were washed once at room temperature in a buffer containing
2 x SSC and 0.1% SDS and twice at 55 C in 0.2 x SSC and
0.5% SDS.
HEK 293 cells were cultured in MEM (Life Technologies, Inc.) supplemented with 10% FBS until approximately 70%
confluent and were then transfected with 4 µg pcDNA3.1/V5/His-tMSTN
and Lipofectamine Plus reagent (Life Technologies, Inc.).
Stable clones were selected for G418 (500 µg/ml) resistance, clonally
derived, and screened via RT-PCR. Stably transfected cells were grown
to confluence and subsequently cultured for 3 days. Conditioned medium
was then concentrated, and buffer was exchanged into PBS by
centrifugation in a Centricon 10 (Amicon, Beverly, MA) spin column and
analyzed by Western blotting. Proteins were separated by reducing PAGE
on 4–12% gradient gels and transferred onto 0.2-µm polyvinylidene
difluoride membranes (all from Novex, San Diego, CA). The
membranes were blocked in 5% nonfat milk prepared in 20 mM
Tris-HCl (pH 7.5), 137 mM NaCl, and 0.1% Tween-20; probed
with 
His (1:1000; Invitrogen) in 5% nonfat milk; and
then probed with horseradish peroxidase-conjugated goat antimouse
antiserum (1:5000; Amersham Pharmacia Biotech, Arlington
Heights, IL) in 20 mM Tris-HCl (pH 7.5), 137 mM
NaCl, and 0.1% Tween-20. Positive immunogenic reactions were
visualized with enhanced chemiluminescence detection reagents
(Amersham Pharmacia Biotech). In addition, the membrane
was stripped (stripping buffer, Chemicon, Temecula, CA), blocked, and
reprobed with V5-horseradish peroxidase as described.
Tissue distribution and developmental expression of tMSTN
Total RNA was isolated from tilapia eye, gill filaments, ovary,
testis, liver, kidney, stomach, gut, brain, heart, skeletal muscle, and
whole larvae as described above and was analyzed by RT-PCR with either
tMSTN forward and reverse primers or with primers specific for
ß-actin (forward, 5'-TAT GGA GAA GAT TTG GCA CC-3'; reverse, 5'-TCA
TCG TAC TCC TGC TTG C-3') or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH; forward, 5'-GCC CCC ATG TTC GTC ATG-3'; reverse, 5'-CTC AGG GAT
GAC CTT GCC-3'). Controls consisted of tilapia genomic DNA (200 ng) and
RNA that was not incubated with Superscript (Life Technologies, Inc.) during the RT reaction. RNA from mouse liver,
kidney, gut, brain, and skeletal muscle as well as from 80–90%
confluent rat L6 myoblasts was similarly analyzed by RT-PCR with either
ß-actin primers or rodent MSTN primers (forward, 5'-GTC TCT CGG ACG
GTA CAT G-3'; reverse, 5'-TGA GCA (C/G)CC (G/A)CA ICG (A/G)TC TAC TAC
C-3'). Similar negative controls were also amplified with these
primers.
First strand cDNA was reverse transcribed from 5 µg total RNA isolated from larvae of different stages of development and analyzed by semiquantitative RT-PCR. After determining that both gene products could be simultaneously amplified in the same tube, the linear portion of the amplification curves for tMSTN and GAPDH were determined by multiplex cycle titration PCR with 0.02 µCi/µl [32P]deoxy-CTP and with cDNA from the earliest larval sample. Aliquots of 5 µl were removed after 25, 30, 35, 40, and 45 cycles of 94 C for 30 sc, 60 C for 30 sec, and 72 C for 1 min and were electrophoresed on 6% polyacrylamide/Tris-HCl, borate, EDTA (TBE) gels. Band intensities were quantified electronically with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). To this end, cDNA from each larval sample was then amplified for 38 cycles, and the tMSTN values were normalized to those of GAPDH.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Furthermore,
the sequences surrounding the initiator codons of both clones satisfy
the minimum Kozak requirements for the initiation of translation
(14).
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). Although the invariant nine cysteine
residues found in all TGFß family members contribute to the formation
of the characteristic cysteine knot through intramolecular disulfide
bonds, it is unknown whether the additional cysteines found in MSTN
orthologs participate in disulfide linkages. However, they are unique
to MSTN and not to other TGFß family members. The estimated mass of
the processed and bioactive carboxyl-terminal peptides for both clones
is approximately 13 kDa.
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, A and B), but are only weakly
homologous to other TGFß family members, excluding
growth/differentiating factor-11 (GDF-11). The degrees of
identity and similarity between the two Perciforme MSTN proteins and
the mammalian MSTN proteins are approximately 62% and 73%,
respectively, vs. 58% and 66% compared with GDF-11. In
addition, white bass MSTN is only 35% similar and 22% identical to a
hybrid striped bass (Morone saxatilis x
chrysops) TGFß ortholog (GenBank Accession No. AF140363).
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). Nevertheless, most of the
dissimilarities with any of the previously described orthologs were
located before the proteolytic processing site. This high level of
conservation suggests that the biological action of MSTN in fish and
possibly in other nonmammalian vertebrates may be equally well
conserved.
Southern, Northern, and Western blotting
Under conditions of low stringency, single bands of 5.5 and 5.0 kb
were recognized by Southern hybridization of a radiolabeled tilapia
cDNA probe to tilapia genomic DNA previously digested with
BamHI and EcoRI, respectively (Fig. 4A). Thus, the tMSTN cDNA sequence
reported herein is probably transcribed from a single gene locus.
Northern hybridization of the same cDNA probe to 15 µg total RNA or 5
µg polyadenylated RNA (data not shown) extracted from adult tilapia
skeletal muscle failed to identify a specific signal. This is in
contrast to previous reports with mice, where MSTN expression was
easily detected by Northern blotting of skeletal muscle RNA.
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). A single band of approximately 13 kDa, consistent
with the predicted mass of the processed and monomeric peptide form,
was detected in conditioned medium from the stably transfected cells,
suggesting that the tilapia protein is appropriately processed and
secreted. The identical band was also recognized by antiserum against
the V5 epitope that lies immediately upstream of the His tag (data not
shown).
tMSTN tissue distribution and developmental expression
pattern
The tissue-specific expression pattern of tMSTN was determined by
RT-PCR (Fig. 5A). In most mammalian
species, MSTN expression is believed to occur primarily in skeletal
muscle and to a much lesser degree in bovine cardiomyocytes and
Purkinje fibers (16). However, tMSTN expression was
detected in a variety of tissues, including the eyes, gill filaments,
ovaries, gut, and brain, but was not present in the liver, kidneys,
stomach, or heart. A minimal amount of tMSTN expression was also
detected in the testis. RNA and cDNA integrity was verified by
denaturing gel electrophoresis and by the successful amplification of
ß-actin in each tissue, respectively. The identification of tMSTN
transcripts in tissues other than skeletal muscle was not due to sample
contamination with genomic DNA, as PCR amplification with primers that
flank the entire open reading frame failed to amplifying a comparable
sized band from 200 ng genomic DNA (Fig. 5B), suggesting a complex
genomic organization. McPherron et al. (9)
determined the MSTN expression pattern in different mouse tissues by
Northern blotting and not by PCR. Therefore, minimal transcript amounts
may have escaped detection in tissues other than skeletal muscle. To
determine whether the differential tissue distribution pattern of MSTN
expression in mouse vs. tilapia tissues might have reflected
differences in the method of detection per se, different
mouse tissues were analyzed by RT-PCR using primers derived from the
mouse MSTN cDNA sequence. In contrast to tilapia, expression of MSTN in
the mouse was confined to skeletal muscle as originally reported (Fig. 5C). Furthermore, it was not detected in L6 myocytes, an immortalized
rat skeletal muscle cell line.
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). Although tMSTN expression was
undetectable in fertilized oocytes and in prehatching larvae with eye
spots (Fig. 6A), it was first detected in posthatching larvae and with
the earliest signs of muscle activity (Fig. 6, B–D). These data
indicate that tMSTN is not constitutively expressed in developing
larvae; rather, its expression appears to be regulated. They also
suggest that tMSTN expression increases with the onset of muscle
activity in developing larvae and, thus, muscle development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although MSTN expression in mouse skeletal muscle is readily detected by Northern blotting, it is not uniform and can be either very low or undetectable in some mouse muscle groups (9, 19). This variability is due to the distribution of different fiber types within individual muscles as the abundance of MSTN message is directly correlated to the presence of myosin heavy chain isoform IIb (19). By contrast, MSTN expression levels in tilapia white skeletal muscle were too low to be detected by Northern blotting. Unlike mammalian skeletal muscle, the distribution of fish axial muscle is homogenous and is segregated by fiber type. Therefore, the apparent difference in basal MSTN expression between mouse and tilapia skeletal muscle may not necessarily be due to differences inherent to each animal model, but may reflect the type of fiber sampled. Although widely expressed in different skeletal muscle fibers, mouse MSTN expression was not detected in L6 myoblasts, an immortalized rat skeletal muscle cell line, by PCR amplification using gene-specific primers. This may be due to the differentiation status of the cells; however, it is also conceivable that myocyte immortality is associated with the loss of MSTN expression and thus the consequential loss of negative growth control in this and possibly other muscle cell lines.
It remains to be determined whether the structurally well conserved novel fish proteins described possess the same biological activities as their mammalian counterparts. However, many other TGFß family members have been identified in various fish species and have been determined to possess relatively well conserved actions (20, 21, 22, 23, 24). The expression of MSTN in tissues other than skeletal muscle suggests that the biological actions of MSTN in tilapia, and possibly in other fishes, may not be limited to skeletal muscle growth inhibition, but may also contribute to the homeostatic growth control of other tissues. The diversity of tissues expressing tMSTN suggests that its actions may be equally diverse and may not necessarily be limited to growth control, assuming that tMSTN receptors are coexpressed with the ligand. Although speculative, tMSTN could potentially regulate some species-specific physiological processes that are unrelated to skeletal muscle growth altogether. For example, tMSTN expression in the gill filaments of this euryhaline teleost suggests that it may participate either directly or indirectly in osmoregulation. Its expression in the brain may suggest that tMSTN helps to coordinate neuronal growth and development, which, unlike in mammals, continues throughout the fish lifecycle. The presence of tMSTN in fish gonads may suggest that it helps to maintain reproductive tissues.
In developing tilapia larvae, tMSTN expression was first detected in posthatched larvae and peaked soon thereafter with the appearance of muscle activity. The development of fish skeletal muscle occurs early and rapidly in the course of larval development (25); thus, the onset of tMSTN expression appears to coincide with early myogenesis. Thomas et al. (26) recently demonstrated that recombinant bovine MSTN inhibits myoblast proliferation in a direct and dose-dependent manner. This effect was mediated by a decrease in the levels and activity of cyclin-dependent kinase-2, an increase in the p21 cyclin-dependent kinase inhibitor, and the hypophosphorylation of retinoblastoma protein, all of which are required for cell cycle withdrawal and myoblast differentiation. The coincidental timing of tMSTN expression with early myogenic events suggests that MSTN's role in regulating mammalian skeletal muscle development may be conserved in fish. During the later stages of larval tilapia development, tMSTN expression was more variable than that which occurred in the early stages and could be due to differential growth rates of individual fish. However, if the distribution of tMSTN is as diverse in larvae as it is in adult fish, the fluctuations in tMSTN expression in the latter stages could be due to a tissue-specific differential expression pattern. Nevertheless, these results suggest that the expression of MSTN in fish is developmentally regulated, which is consistent with the developing mouse (9, 17).
The characteristic double muscling phenotype of MSTN null mice and of domestic cattle that harbor mutant MSTN alleles is indicative of the cytokine’s ability to influence skeletal muscle growth and development in these and possibly other mammalian species. The ability of MSTN to regulate mammalian skeletal muscle differentiation is beginning to be understood; however, additional experiments are required to definitively determine its effects on adult skeletal muscle, regardless of the animal model and whether the different MSTN-expressing tissues of tilapia are sensitive to this cytokine’s influences.
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Acknowledgments |
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Footnotes |
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Received May 22, 2000.
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