Characterization of Fxr1 in Danio rerio; a simple vertebrate model to study costamere development
Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands
* Author for correspondence (e-mail: r.willemsen{at}erasmusmc.nl)
Accepted 17 June 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this paper the suitability of the zebrafish as a model organism to study Fxr1p function during early development is explored. As a first step, we present here the initial characterization of Fxr1p in zebrafish. Fxr1p is present in all the cells from zebrafish embryos from the 2/4-cell stage onward; however, during late development a more tissue-specific distribution is found, with the highest expression in developing muscle. In adult zebrafish, Fxr1p is localized at the myoseptum and in costamere-like granules in skeletal muscle. In the testis, Fxr1p is localized in immature spermatogenic cells and in brain tissue Fxr1p displays a predominantly nuclear staining in neurons throughout the brain. Finally, the different tissue-specific isoforms of Fxr1p are characterized.
Since the functional domains and the expression pattern of Fxr1p in zebrafish are comparable to those in higher vertebrates such as mouse and human, we conclude that the zebrafish is a highly suitable model for functional studies of Fxr1p.
Key words: zebrafish, Danio rerio, Fxr1, Fxr1p, costamere, striated muscle, fragile X syndrome.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Various animal models have been created to study the physiological function
of the three genes. The Fmr1 knockout mice display deficits in visual
spatial performance and have macroorchidism, illustrating similarities between
fragile X patients and this mouse model
(Bakker et al., 1994). In
addition, Fmr1 knockout mice show altered dendritic spine morphology,
indicating a reduced maturation/pruning of spines
(Greenough et al., 2001
).
Fxr2 knockout mice show a mild learning and behaviour phenotype
(Bontekoe et al., 2002
). Thus,
both mouse models point to a mental retardation phenotype in the absence of
Fmrp and Fxr2p, respectively. In contrast, Fxr1 knockout mice die
shortly after birth and show a disruption of the cellular architecture and
structure of both skeletal and cardiac muscle tissue
(Mientjes et al., 2004
). The
absence of Fxr1p in E19 Fxr1 knockout mice results in the
reduced/abnormal expression pattern of costameric proteins like vinculin,
dystrophin and
-actinin and it has been suggested that Fxr1p plays a
role in transport/translational control of structural costameric mRNAs
analogue to FMRP function for dendritic mRNAs
(Mientjes et al., 2004
).
In order to further study the function of FXR1P in the nervous system,
testis and striated muscle tissue, particularly during embryonic development,
it may be advantageous to use a model organism that allows avenues to study
early developmental processes in more detail. The zebrafish Danio
rerio is very suitable for developmental studies as it has a fast
external development, and developing zebrafish remain translucent until the
embryos are free-swimming and organogenesis is complete. Additionally the
availability of techniques to manipulate gene expression, the vast knowledge
base on zebrafish development and the near finished genome project make the
zebrafish an attractive complementary vertebrate model. Importantly, orthologs
of the three FXR genes have been identified in zebrafish
(Wan et al., 2000).
In the present study an initial characterization of Fxr1 in zebrafish has been conducted. We performed sequence analysis, embryonic and adult expression patterns using monospecific antibodies against Fxr1p, and western blotting to detect the different molecular forms of Fxr1p.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dissection of zebrafish
Male fish were euthanized in a 0.2 g l-1 solution of tricaine
(3-amino-benzoic ethylester Sigma, St Louis, MO, USA) and brain, testis and a
strip of dorsal skeletal muscle were dissected, snap frozen in liquid nitrogen
and stored at -80°C till further use.
Fxr-EGFP fusion expression constructs
For total RNA, tissues were removed from -80°C and immediately
homogenized in 1.0 ml trizol. The homogenate was chloroform extracted and RNA
was precipitated according to standard protocols. Subsequently, cDNA was
prepared from 1 µg RNA using AMV RT (Sigma, St Louis, MO, USA) with random
hexamers and oligo dT according to the manufacturer's instructions.
Muscle and brain cDNA was amplified with Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA) using the following Fxr1 primers: f1, 5'-CCGATCGCATGGAGGAACTGACGGTGG-3' and r1, 5'-GTACTCCAGCAGCACCTGTACG-3'. The PCR product was cloned into pCRtopo 2.1 TA (Invitrogen) and, in order to express Fxr1p as an enhanced green fluorescent protein (EGFP) fusionprotein, subcloned into pEGFP-C3 (Clontech, Palo Alto, CA, USA) using the EcoRI sites. Zebrafish Fxr2 cDNA was available as an image clone (GenBank accession number: BC045999), which was ordered from MRC gene service and cloned into pCRtopo 2.1 TA by polymerase chain reaction (PCR) using the primers: f3, 5'-AAGCGACGACGAACATGGAC-3' and r4, 5'-ATGCAAGCAGGGACAGAGTT-3', and subsequently subcloned into pEGFP-C3 (Clontech) using the EcoRI sites. Both constructs were sequence verified.
Primary antibodies
Rabbit monospecific antibodies against Fxr1p were raised according to the
double X program from Eurogentec (Herstal, Belgium). Briefly, synthetic
peptides were produced from the C-terminal amino acid sequence from zebrafish
Fxr1p: AESQSRQTNPRDTRK, and subsequently coupled to keyhole limpet hemocyanin
(KLH). The final bleeding was used to produce an affinity-purified antibody
using affinity purification against the synthetic peptide. The
affinity-purified antibody (named affi 5) was used at a 1:500 dilution for
immunoblotting and 1:25 dilution for immunohistochemistry. Antibodies against
MANDRA1 (mouse anti-dystrophin; Sigma) and vinculin (goat anti-vinculin;
Sanvertech, Santa Cruz, CA, USA) were used for immunohistochemistry at 1:1000
and 1:400 dilution, respectively. Antibodies against P0 and Staufen were from
Immunovision (human; Bereldange, Luxembourg) and Chemicon (rabbit; Temecula,
USA), respectively, and were used at a 1:100 dilution for
immunohistochemistry. The secondary antibodies swine anti-rabbit conjugated
with HRP and rabbit anti-mouse conjugated with horseradish peroxidase (HRP)
were obtained from DAKO (Glostrup, Denmark). Goat anti-rabbit/mouse/human
antibodies conjugated to TRITC or FITC were obtained from Sigma.
Cryosectioning and immunohistochemistry
Adult zebrafish were euthanized (see above) and embedded in Tissue-Tek
(Sakura Finetek Europe BV, Zoeterwoude, The Netherlands). Using a Leica Jung
CM3000 cryostat, 7 µm sections were cut and thaw-mounted on microscopic
slides. Sections were fixed at room temperature for 10 min in 4%
paraformaldehyde in 0.1 mol l-1 Sorrensen buffer, pH 7.3, followed
by a permeabilization step in 100% methanol for 20 min. Sections were rinsed
twice in PBS for 5 min andsubsequent endogenous peroxidase activity was
blocked for those slides that were incubated using the immunoperoxidase
protocol with hydrogen peroxide (0.6%). After blocking, slides were washed
twice in PBS+ (PBS containing 5 g non fat dry milk and 1.5 g glycine
l-1) for 5 min. Incubation with primary antibodies was for 1.5 h at
room temperature or overnight at 4°C. Slides were rinsed three times in
PBS+ for 5 min and incubated with secondary antibodies (both conjugated with
FITC/TRITC or HRP) for 1 h at room temperature. After three washes with PBS+,
slides were either covered with a coverslip using Vectashield containing Dapi
(Vector Laboratories, Burlingame, USA) or further incubated with DAB-substrate
(DAKO) for 6 min, followed by washing in tapwater. Finally, sections were
counterstained using Haematoxylin and embedded in Entellan (Merck, Darmstadt,
Germany). Slides were examined using either a fluorescence microscope or a
bright field microscope.
Cell lines and transfection studies
Cos-1 cells were maintained in DMEM (Gibco Brl, Breda, The Netherlands)
supplemented with 10% foetal calf serum (FCS; Gibco Brl) under 5%
CO2 at 37°C. Cells were seeded on coverslips or in 6-well
plates at 75% confluence the night before transfection. Transient transfection
was performed using Lipofectamine 2000 (Invitrogen) according to the
manufacturer's instructions. Cells were lysed the following day in Ripa (20
mmol l-1 Tris pH7.5, 140 mmol l-1 NaCl, 0.1%
deoxycholate, 0.1% SDS, 0.5% Triton) and complete protease inhibitor cocktail
(Roche, Basel, Switzerland) or, for immunocytochemistry, fixed in 4%
paraformaldehyde in PBS for 10 min and permeabilized in 100% methanol for 20
min. For immunofluorescence the same protocol as for immunohistochemistry was
followed.
Western blotting
Zebrafish tissues were homogenized in Ripa, centrifuged at 12 000
g for 15 min at 4°C and supernatants were stored at
-80°C till further use. Homogenates from zebrafish brain, testis, muscle
and Cos-1 cells transfected with the FXR1-pEGFP or FXR2-pEGFP constructs were
size-separated by 7.5% SDS-PAGE and immunoblotted according to standard
protocols.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Cloning of zebrafish Fxr1
The PCR fragment cloned from zebrafish muscle cDNA spans the open reading
frame and is identical to the published gene bank sequence, except that it
also contains exon 15 (numbering according to
Kirkpatrick et al., 2001;
Fig. 1, boxed region).
Specificity of affinity-purified polyclonal antibody against zebrafish Fxr1p
According to the protein alignment in
Fig. 1 the zebrafish Fxr
proteins are highly homologous and the predicted molecular mass of both Fxr1p
and Fxr2p are approximately identical. The Affi5 antibody was raised against
the zebrafish Fxr1p using a synthetic peptide; however, part of the used
peptide is also present in zebrafish Fxr2p. Therefore we examined whether
Affi5 crossreacts with zebrafish Fxr2p. In order to determine the specificity
of Affi5 we transiently overexpressed zebrafish Fxr1-EGFP and
Fxr2-EGFP in Cos-1 cells and performed immunofluorescence using Affi5
antibody. In addition, cell homogenates were prepared for western blotting.
For immunofluorescence, Affi5 showed a strong labelling of Cos-1 cells
expressing Fxr1p-EGFP (Fig. 2A
for GFP staining and 2B for Affi5 staining), whereas Cos-1 cells
overexpressing Fxr2p-EGFP showed total absence of labelling for Affi5
(Fig. 2D) while an intense
staining could be detected for GFP fluorescence signal
(Fig. 2C).
|
In immunoblotting, Affi5 recognizes the Fxr1p-EGFP fusion protein. The size of the band is approximately 100 kDa, which reflects a molecular mass of 73 kDa for Fxr1p plus 27 kDa for EGFP. In contrast, Affi5 staining did not detect the Fxr2p-EGFP fusion protein (Fig. 2F, compare lanes 1 and 2). Note that equal amounts of Fxr1-EGFP and Fxr2-EGFP fusion protein were present as shown by immunoblotting using antibodies against GFP (Fig. 2E, lanes 1 and 2).
Distribution of Fxr1p in adult zebrafish
In man and in mouse Fxr1p is highly expressed in skeletal muscle, heart,
testis and brain. As an indication whether Fxr1p has a role in zebrafish
comparable to that in mouse or in human, and whether it would therefore be a
suitable model to study the function of Fxr1p, we examined the distribution of
Fxr1p in the zebrafish using Affi5 antibodies in cryostat sections from adult
zebrafish.
Immunoreactivity of Affi5 was observed in the brain, most notably in the Purkinje cells of the cerebellum and a number of neurons in the brainstem. Surprisingly, a significant number of neurons throughout the brain display nuclear staining (Fig. 3A).
|
Although Fxr1p is highly expressed in the testis, the signal is restricted to the immature spermatogenic cells (Fig. 3B). Skeletal muscle showed the highest level of Fxr1p expression, where it was localized in granular structures throughout the muscle fiber and intensely at the sarcolemma. Furthermore, a very intense and granular staining was observed bordering the myoseptum (Fig. 3C,D). The signal of Fxr1p at the myoseptum extends somewhat into the cytoplasm, more so than at the sarcolemma.
Fxr1p in the developing zebrafish
To study the expression of Fxr1p during embryonic development and to test
whether it was localized in a similar pattern as in adult tissues, zebrafish
embryos at different stages were embedded in Tissue Tek and cryosections were
immunoincubated with Affi5.
Fxr1p is already detectable at the 2/4 cell stage, where it is distributed evenly over the cell mass (data not shown). At the dome/epiboly stage at 6 h.p.f. (hours post fertilization), Fxr1p is present at high levels in all the cells (Fig. 4A). From early somitogenesis onward Fxr1p is expressed at very high levels in myoblasts throughout the somites. During the maturation of the embryos (1-5 d.p.f.) the immunoreactivity gradually concentrates at the myosepta and at the sarcolemma in regularly placed granular structures. Fig. 4B illustrates the weak Fxr1p expression in the head of an embryo 1 d.p.f.,whereas the tail from 1 d.p.f. embryos showed a high Fxr1p expression in myoblasts (Fig. 4C). In embryos at 3 d.p.f. Fxr1p expression level is moderate in the brain (Fig. 4D) and very high in myoblasts (Fig. 4E).
|
Fxr1p isoforms are differentially expressed in zebrafish tissues.
In the mouse, several different isoforms of Fxr1p have been described, due
to extensive alternative splicing. We examined the presence of different
isoforms of Fxr1p in zebrafish by western blot analysis using different
tissues, including brain, skeletal muscle and testis. In brain, the most
prominent isoform is approximately 74 kDa
(Fig. 5, lane 1). In muscle
tissue we could detect Fxr1p isoforms (two major bands) of approximately 80-88
kDa (Fig. 5, lane 2) and in
testis the most prominent isoform was 72 kDa
(Fig. 5, lane 3).
|
Colocalization of Fxr1p with components of the translational machinery
In mammals, Fxr1p is incorporated in mRNP particles within actively
translating ribosomes (Ceman et al.,
1999; Dube et al.,
2000
; Khandjian et al.,
1998
; Tamanini et al.,
1999
). Furthermore, it has been described that (poly)ribosomes are
located at the myoseptum, and that transcripts can be translated locally
(Horne and Hesketh, 1990
;
Ovalle, 1987
). We therefore
set out to determine whether P0, a protein component of ribosomes, and
Staufen, which can form complexes with both FMRP and FXR1P and is known to be
involved in transport/translation of mRNAs, are also localized at the
myoseptum.
To this end, cryosections of adult zebrafish were immunoincubated simultaneously with Affi5 in combination with anti-Staufen or anti-P0 antibodies. Both the Staufen antibody and the P0 antibody recognize the zebrafish orthologs and show a strong immunoreactivity at the myoseptum and at the sarcolemma. Staufen immunoreactivity appears to be more concentrated around junctions of fibers (Fig. 6A), whereas P0 appears to be relatively more localized at the myoseptum (Fig. 6E). Simultaneous distribution with Fxr1p (Fig. 6B,D) illustrates the colocalization with Staufen to some extent (Fig. 6C, merge) and with P0 in higher quantities (Fig. 6F, merge).
|
Fxr1p is localized next to dystrophin and vinculin at the myoseptum
The localization of Fxr1p at the myoseptum is reminiscent of that of
dystrophin in zebrafish. Additionally, Fxr1p has been reported to be localized
in costameres (Dube et al.,
2000). In order to study whether Fxr1p is colocalized with
vinculin and dystrophin, two proteins of the costameric protein network, and
to explore a potential role of Fxr1p in the maintenance of the structural
integrity of costamers, we examined the localization of Fxr1p in relation to
dystrophin by double immunofluorescence in combination with confocal
microscopy using cryosections from muscle tissue.
Frx1p, vinculin and dystrophin are all three localized at the myoseptum. However, this close localization at the myoseptum of Fxr1p on the one hand and vinculin and dystrophin on the other is not an exact colocalization, as shown in Fig. 7 using confocal analysis. Closer examination reveals that the signals only partly overlap. Both vinculin and dystrophin are localized more to the centre of the myoseptum than is Fxr1p.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To examine the feasibility of the zebrafish as a model for Fxr1p function we first compared the zebrafish Fxr1p sequence with that from human and mouse. Fxr1p is highly conserved between these different species and all major domains that have been described to play a role in the function of the FXR family of proteins are present in zebrafish Fxr1p, although the alignment for the RGG-box domain in zebrafish Fxr1p was not unambiguously clear (Fig. 1). Nevertheless, the evolutionarily conserved domains in zebrafish Fxr1p suggest that Fxr1p has a cellular function in zebrafish similar to that in human and mouse.
Next we examined the expression pattern of Fxr1p in zebrafish to establish
whether it is expressed predominantly in skeletal muscle, testis and brain, as
in mammals. In embryos, Fxr1p is ubiquitously expressed in all cells between 0
h.p.f. and 6 h.p.f. From 1 d.p.f. onward, Fxr1p showed a more tissue-specific
expression with a very high expression in myoblasts and a moderate expression
level in neurons from the central nervous system. This more differential
expression was also observed during late embryonic development in the mouse
(De Diego Otero et al., 2000).
In 3 d.p.f. embryos, Fxr1p was present in almost all the neurons of the
central nervous system with a high expression in the Purkinje cells of the
cerebellum, but, surprisingly, a significant number of neurons displayed a
predominantly nuclear staining. Interestingly, a nuclear staining of neurons
has also been reported for FXR1P in human foetal brain (18 weeks;
Tamanini et al., 1997
).
The labelling intensity of Fxr1p in skeletal muscle tissue from both 1 and
3 d.p.f. embryos was very high compared to the brain tissue and suggests an
important role for Fxr1p in myogenesis. At this stage of development Fxr1p
already showed the characteristic costamere localization. In adult zebrafish,
Fxr1p expression was tissue-specific and similar to the differential
expression in man and mouse, that is, high expression in brain, striated
muscle tissue and testis. However, the subcellular distribution of Fxr1p in
neurons from adult zebrafish was predominantly nuclear as also observed in 3
d.p.f. embryos. This contrasts with the human and mouse subcellular Fxr1p
localization in neurons, which is predominantly cytoplasmic
(Bakker et al., 2000;
Khandjian et al., 1998
). The
difference in subcellular localization of Fxr1p indicates that the cellular
context of adult zebrafish neurons may share characteristics with that of
human foetal neurons (Tamanini et al.,
1997
).
The subcellular localization of Fxr1p in striated muscle tissue was in
granular structures at the sarcolemma, which appear to be costameres as the
granular Fxr1p staining overlaps with that of vinculin and dystrophin, both
are components of the costameric protein network
(Morris and Fulton, 1994;
Bassett et al., 2003
;
Bassett and Currie, 2003
;
Costa et al., 2003
). This is
in agreement with previous reports in mice that also described Fxr1p staining
in granular structures in costameres (Dube
et al., 2000
; Mientjes et al.,
2004
). Most striking is the predominant localization of Fxr1p at
the myosepta. These structures have been linked to laminar tendons and serve
to transmit the force of the contracting muscle segments to the vertebral
column (Gemballa and Roder,
2004
; Gemballa and Vogel,
2002
). This localization of Fxr1p is, however, not entirely
surprising as Fxr1p is probably, like FMR1P, involved in transport and/or
regulation of translation of specific mRNAs. It has been described that both
at the myoseptum and next to the costameres large numbers of actively
transcribing (poly)ribosomes are located
(Morris and Fulton, 1994
).
Considering these findings it is tempting to hypothesize that Fxr1p is
involved in local translation of transcripts encoding proteins that are of
importance for these structures. Further exploring this notion, we determined
the localization of Staufen and P0 in double-labelling experiments. Indeed,
both proteins showed a colocalization with Fxr1p, albeit with different
intensities. The presence of P0, a component of the 60S ribosomal precursor
unit, illustrates the presence of ribosomes at the myoseptum and at the
sarcolemma in zebrafish skeletal muscle. Thus, Fxr1p might be associated to
(poly)ribosomes in zebrafish muscle, which is in line with a role for Fxr1p in
transport and/or translation of specific mRNAs in the vicinity of costameres
(Morris and Fulton, 1994
).
Staufen has been reported to be present in RNP particles that also contain
FMR1 and FXR1p (Ohashi et al.,
2002). Recent data show that Staufen protein is localized at the
neuromuscular junctions (NMJ) and may be involved in maturation and plasticity
of the NMJ (Belanger et al.,
2003
). Although Staufen is, like Fxr1p, present both at the
sarcolemma and at the myoseptum in zebrafish muscle, it has a distinctly
different pattern of signal intensity, being more concentrated around
junctions of fibers with the myoseptum and other muscle fibers. These
concentrations of Staufen immunoreactivity could correspond to the NMJs. This
suggests that, although Fxr1p and Staufen can both be present in RNP particles
in the brain and may partially colocalize in zebrafish muscle, both proteins
have distinct roles in zebrafish skeletal muscle tissue.
The localization of Fxr1p at the myoseptum is reminiscent of the
localization of dystrophin and vinculin in zebrafish. Both proteins are
components of the myoseptum and provide a connection between the extracellular
matrix (ECM) and the intracellular cytoskeleton
(Bassett et al., 2003;
Bassett and Currie, 2003
;
Costa et al., 2003
).
Hypothetically, Fxr1p could be involved in maintaining muscle fiber
integrity by a direct binding to components of the myoseptum, such as
dystrophin or vinculin. We therefore examined the possible colocalization of
dystrophin and vinculin protein using confocal immunofluorescence imaging. As
both vinculin and dystrophin are distinctly more centrally localized at the
myoseptum than Fxr1p, it is unlikely that Fxr1p is part of the
dystrophin-containing complex that anchors the muscle fiber to the ECM.
However, dystrophin mRNA shows a distinct localization bordering at the
myoseptum from 19 h.p.f. onward and appears to be located immediately outside
the myoseptum itself, where dystrophin protein is located
(Bassett et al., 2003).
Comparing these findings to our confocal study on the possible colocalization
of Fxr1p with dystrophin or vinculin, it appears likely that Fxr1p may
colocalize with dystrophin mRNA. Further in situ hybridisation
studies are necessary to establish this colocalization.
The Fxr1p localization in zebrafish testis appears to be predominantly in
all the immature spermatogenic cells, which has also been observed in mouse
and human, albeit Fxr1p immunoreactivity has also been reported in the tails
of murine sperm using antibodies against high molecular isoforms of Fxr1p
(Huot et al., 2001;
Tamanini et al., 1997
).
In conclusion, the functional domains of Fxr1p are evolutionary conserved in zebrafish and the expression pattern of zebrafish Fxr1p is consistent with the expression of the Fxr1p orthologs in mouse and man. Thus, zebrafish should be an outstanding model organism to study the cellular function of Fxr1p, particularly during embryonic development and neonatally. Gene knockdown experiments using the morpholino gene-targeting strategy and transgenic techniques using expression plasmids with Fxr1-EGFP especially may open new avenues that will lead to knowledge about the in vivo function of Fxr1p.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashley, C., Jr, Wilkinson, K. D., Reines, D. and Warren, S. T. (1993). FMR1 protein: conserved RNP family domains and selective RNA binding. Science 262,563 -568.[Medline]
Bakker, C. E., de Diego Otero, Y., Bontekoe, C., Raghoe, P., Luteijn, T., Hoogeveen, A. T., Oostra, B. A. and Willemsen, R. (2000). Immunocytochemical and biochemical characterization of FMRP, FXR1P, and FXR2P in the mouse. Exp. Cell Res. 258,162 -170.[CrossRef][Medline]
Bakker, C. E., Verheij, C., Willemsen, R., Vanderhelm, R., Oerlemans, F., Vermey, M., Bygrave, A., Hoogeveen, A. T., Oostra, B. A., Reyniers, E. et al. (1994). Fmr1 knockout mice: A model to study fragile X mental retardation. Cell 78, 23-33.[Medline]
Bassett, D. I., Bryson-Richardson, R. J., Daggett, D. F.,
Gautier, P., Keenan, D. G. and Currie, P. D. (2003).
Dystrophin is required for the formation of stable muscle attachments in the
zebrafish embryo. Development
130,5851
-5860.
Bassett, D. I. and Currie, P. D. (2003). The
zebrafish as a model for muscular dystrophy and congenital myopathy.
Hum. Mol. Genet. 12 Special
issue 2, R265-R270.
Belanger, G., Stocksley, M. A., Vandromme, M., Schaeffer, L., Furic, L., DesGroseillers, L. and Jasmin, B. J. (2003). Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction. J. Neurochem. 86,669 -677.[CrossRef][Medline]
Bontekoe, C. J., McIlwain, K. L., Nieuwenhuizen, I. M., Yuva-Paylor, L. A., Nellis, A., Willemsen, R., Fang, Z., Kirkpatrick, L., Bakker, C. E., McAninch, R. et al. (2002). Knockout mouse model for Fxr2: a model for mental retardation. Hum. Mol. Genet. 11,487 -498.[CrossRef][Medline]
Briggs, J. P. (2002). The zebrafish: a new model organism for integrative physiology. Am. J. Physiol. 282,R3 -R9.
Brown, V., Jin, P., Ceman, S., Darnell, J. C., O'Donnell, W. T., Tenenbaum, S. A., Jin, X., Feng, Y., Wilkinson, K. D., Keene, J. D. et al. (2001). Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in Fragile X Syndrome. Cell 107,477 -487.[Medline]
Ceman, S., Brown, V. and Warren, S. T. (1999).
Isolation of an FMRP-associated messenger ribonucleoprotein particle and
identification of nucleolin and the Fragile X-related proteins as components
of the complex. Mol. Cell. Biol.
19,7925
-7932.
Costa, M. L., Escaleira, R., Manasfi, M., de Souza, L. F. and Mermelstein, C. S. (2003). Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis. Braz. J. Med. Biol. Res. 36,1117 -1120.[Medline]
Darnell, J. C., Jensen, K. B., Jin, P., Brown, V., Warren, S. T. and Darnell, R. B. (2001). Fragile X mental retardation protein targets g quartet mRNAs important for neuronal function. Cell 107,489 -499.[Medline]
Darnell, R. B. (2004). Paraneoplastic
neurologic disorders: windows into neuronal function and tumor immunity.
Arch. Neurol. 61,30
-32.
De Diego Otero, Y., Bakker, C. E., Raghoe, P., Severijnen, L. W. F. M., Hoogeveen, A., Oostra, B. A. and Willemsen, R. (2000). Immunocytochemical characterization of FMRP, FXR1P and FXR2P during embryonic development in the mouse. Gene Funct. Dis. 1, 28-37.
De Diego Otero, Y., Severijnen, L. A., van Cappellen, G.,
Schrier, M., Oostra, B. and Willemsen, R. (2002). Transport
of fragile X mental retardation protein via granules in neurites of PC12
cells. Mol. Cell Biol.
22,8332
-8341.
Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P. and Mandel, J. L. (1993). The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation. Nature Genet. 4,335 -340.[Medline]
Dodd, A., Curtis, P. M., Williams, L. C. and Love, D. R.
(2000). Zebrafish: bridging the gap between development and
disease. Hum. Mol. Genet.
9,2443
-2449.
Dooley, K. and Zon, L. I. (2000). Zebrafish: a model system for the study of human disease. Curr. Opin. Genet. Dev. 10,252 -256.[CrossRef][Medline]
Dube, M., Huot, M. E. and Khandjian, E. W. (2000). Muscle specific Fragile X related protein 1 isoforms are sequestered in the nucleus of undifferentiated myoblast. BMC Genet. 1,1 -4.[Medline]
Eberhart, D. E., Malter, H. E., Feng, Y. and Warren, S. T.
(1996). The fragile X mental retardation protein is a
ribosonucleoprotein containing both nuclear localization and nuclear export
signals. Hum. Mol. Genet.
5,1083
-1091.
Fridell, R. A., Benson, R. E., Hua, J., Bogerd, H. P. and Cullen, B. R. (1996). A nuclear role for the fragile X mental retardation protein. EMBO J. 15,5408 -5414.[Abstract]
Gemballa, S. and Roder, K. (2004). From head to tail: the myoseptal system in basal actinopterygians. J. Morphol. 259,155 -171.[CrossRef][Medline]
Gemballa, S. and Vogel, F. (2002). Spatial arrangement of white muscle fibers and myoseptal tendons in fishes. Comp. Biochem. Physiol. 133A,1013 -1037.
Greenough, W. T., Klintsova, A. Y., Irwin, S. A., Galvez, R.,
Bates, K. E. and Weiler, I. J. (2001). Synaptic regulation of
protein synthesis and the fragile X protein. Proc. Natl. Acad. Sci.
USA 98,7101
-7106.
Haffter, P., Granato, M., Brand, M., Mullins, M. C.,
Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J.,
Heisenberg, C. P. et al. (1996). The identification of genes
with unique and essential functions in the development of the zebrafish,
Danio rerio. Development
123, 1-36.
Horne, Z. and Hesketh, J. (1990). Immunological localization of ribosomes in striated rat muscle. Evidence for myofibrillar association and ontological changes in the subsarcolemmal:myofibrillar distribution. Biochem. J. 268,231 -236.[Medline]
Huot, M. E., Mazroui, R., Leclerc, P. and Khandjian, E. W.
(2001). Developmental expression of the fragile X-related 1
proteins in mouse testis: association with microtubule elements.
Hum. Mol. Genet. 10,2803
-2811.
Khandjian, E. W., Bardoni, B., Corbin, F., Sittler, A., Giroux,
S., Heitz, D., Tremblay, S., Pinset, C., Montarras, D., Rousseau, F. et
al. (1998). Novel isoforms of the fragile X related protein
FXR1P are expressed during myogenesis. Hum. Mol.
Genet. 7,2121
-2128.
Kirkpatrick, L. L., McIlwain, K. A. and Nelson, D. L. (2001). Comparative genomic sequence analysis of the FXR gene family: FMR1, FXR1, and FXR2. Genomics 78,169 -177.[CrossRef][Medline]
Laggerbauer, B., Ostareck, D., Keidel, E. M., Ostareck-Lederer,
A. and Fischer, U. (2001). Evidence that fragile X mental
retardation protein is a negative regulator of translation. Hum.
Mol. Genet. 10,329
-338.
Mientjes, E. J., Willemsen, R., Kirkpatrick, L. L.,
Nieuwenhuizen, I. M., Hoogeveen-Westerveld, M., Vermeij, M., Reis, S.,
Bardoni, B., Hoogeveen, A. T., Oostra, B. A. et al. (2004).
Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p
function in vivo. Hum. Mol. Genet.
13,1291
-1302.
Morris, E. J. and Fulton, A. B. (1994).
Rearrangement of mRNAs for costamere proteins during costamere development in
cultured skeletal muscle from chicken. J. Cell Sci.
107,377
-386.
Ohashi, S., Koike, K., Omori, A., Ichinose, S., Ohara, S.,
Kobayashi, S., Sato, T. A. and Anzai, K. (2002).
Identification of mRNP complexes containing pur alpha, mStaufen, fragile X
protein and myosin Va, and their association with rough endoplasmic reticulum
equipped with a kinesin motor. J. Biol. Chem.
277,37804
-37810.
Oostra, B. A. and Willemsen, R. (2003). A
fragile balance: FMR1 expression levels. Hum. Mol.
Genet. 12 Special issue 2,R249
-R257.
Ovalle, W. K. (1987). The human muscle-tendon junction. A morphological study during normal growth and at maturity. Anat. Embryol. Berlin 176,281 -294.[Medline]
Schaeffer, C., Bardoni, B., Mandel, J. L., Ehresmann, B.,
Ehresmann, C. and Moine, H. (2001). The fragile X mental
retardation protein binds specifically to its mRNA via a purine quartet motif.
EMBO J. 20,4803
-4813.
Siomi, H. and Dreyfuss, G. (1997). RNA-binding proteins as regulators of gene expression. Curr. Opin. Genet. Dev. 7,345 -353.[CrossRef][Medline]
Siomi, H., Matunis, M. J., Michael, W. M. and Dreyfuss, G. (1993a). The pre-mRNA binding K protein contains a novel evolutionarily conserved motif. Nucleic Acids Res. 21,1193 -1198.[Abstract]
Siomi, H., Siomi, M. C., Nussbaum, R. L. and Dreyfuss, G. (1993b). The protein product of the fragile X gene, FMR1, has characteristics of an RNA-binding protein. Cell 74,291 -298.[Medline]
Sittler, A., Devys, D., Weber, C. and Mandel, J.-L.
(1996). Alternative splicing of exon 14 determines nuclear or
cytoplasmic localisation of FMR1 protein isoforms. Hum. Mol.
Genet. 5,95
-102.
Strausberg, R. L., Feingold, E. A., Grouse, L. H., Derge, J. G.,
Klausner, R. D., Collins, F. S., Wagner, L., Shenmen, C. M., Schuler, G. D.,
Altschul, S. F. et al. (2002). Generation and initial
analysis of more than 15,000 full-length human and mouse cDNA sequences.
Proc. Natl. Acad. Sci. USA
99,16899
-16903.
Tamanini, F., Van Unen, L., Bakker, C., Sacchi, N., Galjaard, H., Oostra, B. A. and Hoogeveen, A. T. (1999). Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P. Biochem. J. 343,517 -523.[CrossRef][Medline]
Tamanini, F., Willemsen, R., van Unen, L., Bontekoe, C.,
Galjaard, H., Oostra, B. A. and Hoogeveen, A. T. (1997).
Differential expression of FMR1, FXR1 and FXR2 proteins in human brain and
testis. Hum. Mol. Genet.
6,1315
-1322.
Wan, L., Dockendorff, T. C., Jongens, T. A. and Dreyfuss, G.
(2000). Characterization of dFMR1, a Drosophila
melanogaster homolog of the Fragile X mental retardation protein.
Mol. Cell. Biol. 20,8536
-8547.
Willemsen, R., Oostra, B. A., Bassell, G. J. and Dictenberg, J. (2004). The fragile X syndrome: From molecular genetics to neurobiology. Ment. Retard. Dev. Disabil. Res. Rev. 10, 60-67.[CrossRef][Medline]
Zalfa, F., Giorgi, M., Primerano, B., Moro, A., Di Penta, A., Reis, S., Oostra, B. and Bagni, C. (2003). The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses. Cell 112,317 -327.[Medline]
Zhang, Y., Oconnor, J. P., Siomi, M. C., Srinivasan, S., Dutra, A., Nussbaum, R. L. and Dreyfuss, G. (1995). The fragile X mental retardation syndrome protein interacts with novel homologs FXR1 and FXR2. EMBO J. 14,5358 -5366.[Abstract]