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First published online 31 January 2007
doi: 10.1242/dev.02783
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1 Institute of Developmental Biology and Molecular Medicine, School of Life
Science, Fudan University, Shanghai, 200433, China.
2 Institute of Neuroscience and Key Laboratory of Neurobiology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai,
200031, China.
3 Howard Hughes Medical Institute and Department of Genetics, Yale University
School of Medicine, New Haven, CT 06520, USA.
4 Department of Immunology, Duke University Medical Center, Durham, NC 27706,
USA.
5 Howard Hughes Medical Institute and Department of MCDB, University of
Colorado, Boulder, CO 80309, USA.
* Author for correspondence (e-mail: rener_xu{at}fudan.edu.cn)
Accepted 14 December 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Synaptic nuclei, Neuromuscular junction, Neonatal lethality, Nuclear envelope, KASH, SUN domain, Nesprin, ANC-1, MSP-300, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Starr and Han, 2003). In
recent years, genetic analyses in several model organisms have shown that the
Klarsicht/ANC-1/Syne homologue (KASH)-domain-containing proteins that are
associated with the nuclear envelope play important roles in nuclear
positioning during various cellular and developmental processes
(Grady et al., 2005;
Malone et al., 2003;
Mosley-Bishop et al., 1999;
Starr and Han, 2002;
Starr et al., 2001;
Yu et al., 2006).
The KASH domain is a conserved protein motif of approximately 60 amino
acids that is located at the C-terminus of KASH-family proteins
(Starr and Fischer, 2005).
KASH domains have been shown to bind to nuclear envelope and are likely to be
responsible for the association of the nuclear envelope with KASH proteins
(Fischer et al., 2004;
Grady et al., 2005;
Malone et al., 2003;
Starr and Han, 2002;
Wilhelmsen et al., 2005;
Yu et al., 2006;
Zhang et al., 2001;
Zhen et al., 2002). In C.
elegans, the KASH-domain proteins UNC-83, ANC-1 and ZYG-12 have been
shown to be associated with the nuclear envelope and to play roles in nuclear
migration, nuclear anchorage of syncytial cells and association of centrosomes
with the nuclear envelope during cell division, respectively
(Hedgecock and Thomson, 1982;
Horvitz and Sulston, 1980;
Malone et al., 2003;
Starr and Han, 2002;
Starr et al., 2001). In
Drosophila, the KASH-domain protein Klarsicht has been shown to be
important for nuclear migration during eye development and for the movement of
lipid droplets (Mosley-Bishop et al.,
1999; Welte et al.,
1998). The Drosophila MSP-300, a homologue of the worm
ANC-1 protein in overall structure (Starr
and Han, 2002; Volk,
1992; Zhang et al.,
2002), was also shown to be associated with the nuclear envelope
and to play a crucial role in anchoring nurse-cell nuclei during oogenesis
(Yu et al., 2006).
Three KASH-domain-containing proteins have been discovered in mammals,
namely Syne-1 (also known as Syne1, Myne1, Nesprin-1, Enaptin165), Syne-2
(also known as Syne2 and Nesprin-2; and as NUANCE in humans) and Nesprin-3
(Apel et al., 2000;
Gough et al., 2003;
Mislow et al., 2002;
Padmakumar et al., 2004;
Wilhelmsen et al., 2005;
Zhang et al., 2001;
Zhen et al., 2002). Nesprin-3
is a much smaller protein compared to the other two Syne proteins. Syne-1 and
Syne-2 are orthologs of ANC-1 and MSP-300: all four proteins are very large
(>6000 amino acids) and contain actin-binding domains at their N-terminus,
a large middle part and a KASH domain at their C-terminus
(Starr and Fischer, 2005). The
KASH domains of Syne proteins have been shown to target proteins to the
nuclear envelope (Apel et al.,
2000; Grady et al.,
2005; Zhang et al.,
2001; Zhen et al.,
2002). The structural similarities of Syne proteins to ANC-1 and
MSP-300 suggest that they may also be involved in nuclear anchorage during
important cellular and developmental processes.
Syne proteins have been implicated in playing important roles in nuclear
positioning in multinucleated skeletal muscle cells. During early development
of the skeletal muscle, hundreds of myoblasts fuse together to form
multinucleated myotubes, and nuclei undergo migration
(Englander and Rubin, 1987).
Later on, each myotube matures into a large syncytial muscle fiber and nuclei
are stably anchored at the periphery of each individual cell
(Bruusgaard et al., 2003).
Noticeably, except for a 3-8 nuclei (synaptic nuclei) cluster under the
neuromuscular junction (NMJ), myonuclei distribute evenly in muscle fibers
(Sanes and Lichtman, 1999).
The evenly spaced localization pattern of non-synaptic nuclei is speculated to
result from nuclei repelling each other to minimize the transport distance
(Bruusgaard et al., 2003).
However, the underlying mechanism responsible for nuclear anchorage remains
unknown. In addition, synaptic nuclei have long been proposed to be
transcriptionally specialized and essential in maintaining the postsynaptic
components of the NMJ (Sanes and Lichtman,
2001; Schaeffer et al.,
2001), but the anchoring mechanism for those nuclei has also been
obscure. Both Syne-1 and Syne-2 have been found to be expressed at high levels
in the skeletal muscle (Apel et al.,
2000; Zhang et al.,
2005; Zhang et al.,
2001). More recently, a direct involvement of Syne proteins in
nuclear positioning has been indicated in the study of transgenic mice
expressing a dominant-negative form of Syne-1
(Grady et al., 2005). This
study showed that the ectopic expression of the dominant-negative form of
Syne-1 disrupted the positioning of synaptic nuclei but had no effect on the
even spacing of non-synaptic nuclei. However, it remains to be determined to
what degree this dominant-negative effect reflects the function of Syne
proteins and which Syne protein is directly involved in myonuclear positioning
during muscle development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genomic DNA was extracted and genotyped using a three-primer PCR. Primers for Syne-1 genotyping were prcmy016, prcmy017 and prcmy018 (sequence details of all primers mentioned in this paper are available upon request). Primers for Syne-2 genotyping were prcmy019, prcmy020 and prcmy021.
Southern blot was carried out according to standard protocols
(Sambrook and Russell, 2001).
For Syne-1, genomic DNA samples were digested with BamHI and
hybridized with a probe obtained from the PCR amplification of C57/B6J genomic
DNA with the primers XP141 and XP142. For Syne-2, genomic DNA samples
were digested by SpeI and hybridized with a probe obtained from the
PCR amplification of C57/B6J genomic DNA with the primers XP154 and XP155.
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Preparation of antibodies against Syne-1, Syne-2 and SUN2
DNA fragments from cDNA clones (ATCC) of Syne-1, Syne-2 and
SUN2 were cloned into pET28 or pET32 after PCR amplification with the
following primers: prDX059 and prDX060 for the 115 amino acid peptide at the
C-terminus of Syne-1; prDX061 and prDX062 for the 66 amino acid peptide at the
C-terminus of Syne-2; and prDX057 and prDX058 for the 123 amino acid peptide
of SUN2. Production of recombinant proteins was induced using IPTG in E.
coli BL21 and proteins were purified using a Niagarose column. Polyclonal
antibodies were produced by immunizing rabbits with the purified 6xHis
fusion proteins, and the rabbit anti-sera were affinity-purified with HiTrap
NHS-activated HP columns (Amersham Bioscience).
Histological analysis
For frozen tissue sections, tissues of interest were dissected out,
embedded in OCT and frozen in liquid-nitrogen-cooled isopentane. Sections (6-8
µm) were then collected.
For paraffin sections, embryos or tissues of interest were dissected out, fixed in 4% formaldehyde, dehydrated in ethanol, cleared with xylene and embedded in paraffin. Sections (5 µm) were then collected and were stained with hematoxylin and Eosin.
Immunofluorescence staining and microscopy
To analyze myonuclei of adult mice, whole-mount staining of the tibialis
anterior (leg muscle) was carried out following the protocol described
previously (Grady et al.,
2005). The myc tag was labeled with 9E10-FITC (Sigma).
Skeletal muscle fibers of E18.5 embryos were analyzed by fixing their
thoraci and then dissecting out their triangularis sterni, which were then
stained with tetramethylrhodamine-conjugated 
-bungarotoxin (BTX)
(Molecular probes) and DAPI diluted in blocking solution (PBS containing 2%
goat serum and 0.4% Triton). After thorough washing, individual fibers were
teased out, mounted in mounting medium (Vectashield) and viewed under a light
microscope (Misgeld et al.,
2002).
To analyze branches of the phrenic nerve, diaphragms were dissected out, fixed and stained with a mixture of rabbit anti-neurofilament (Chemicon) and anti-synaptophysin (Zymed). The muscles were then washed and incubated with goat anti-rabbit IgG-FITC (Sigma), BTX and DAPI. After extensive washing, diaphragms were mounted and viewed under either a light microscope (for low magnification) or a confocal microscope for higher-resolution pictures (Leica).
Immunohistology of frozen tissue sections was carried out following
standard protocols (Harlow and Lane,
1999). The following commercial antibodies were used: MuSK
(Sigma), rapsyn (Sigma), synaptophysin (Zymed) and utrophin (Novocastra).
Electrophysiology
Diaphragms with intact phrenic nerves were isolated from E18.5 embryos and
balanced in an oxygenated solution containing 125 mM NaCl, 2.5 mM KCl, 2 mM
CaCl2, 12 mM MgCl2, 1.3 mM
NaH2PO4, 25 mM NaHCO3 and 10 mM glucose (pH
7.3) at room temperature. The phrenic nerve was stimulated through a suction
electrode, and the diaphragm was penetrated near the main intramuscular nerve.
Intracellular recordings were made with microelectrodes that measured 20-70
megaohms when filled with 3 M KCl. All data were digitized at 10 kHz and
collected on magnetic disks with Axotape software (Axon Instruments).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To further confirm that the KASH domains were eliminated in these mutants, we first carried out reverse transcriptase (RT)-PCR with total mRNA from both heart and skeletal muscle, and found that the coding sequence for each KASH domain could not be amplified, whereas a middle segment of each gene was transcribed (data not shown). Secondly, we used immunofluorescence staining to examine nuclear envelope-localized Syne proteins with antibodies against the C-terminal part of each protein (see Materials and methods). Both Syne-1 and Syne-2 were found to localize to the nuclear envelope in heterozygous skeletal muscle but not in homozygous-mutant mice (Fig. 1E-H). We concluded that we had established Syne-1 and Syne-2 KASH-domain-knockout mice, referred to as Syne-1-/- and Syne-2-/- mice, respectively.
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), but the dominant-negative effect of the transgene was
not sufficient to eliminate the endogenous Syne-1 protein from the nuclear
envelope. We thus analyzed nuclei positioning in syncytial skeletal muscle
cells in Syne-1-/- mice. In order to exclude the
interference of nuclei from connective tissues and Schwann cells outside
muscle cells when DAPI staining was used, we employed an anti-SUN2 antibody as
an additional marker to label myonuclei, as the antibody specifically
recognizes the nuclear envelope of skeletal muscle cells
(Fig. 2A-C, and see text below;
unpublished data from X.D.). In wild-type mice, non-synaptic nuclei distribute uniformly along the whole muscle fiber (Fig. 2A). By marked contrast, nuclei clustered together abnormally in Syne-1-/- mice (a nuclear cluster was defined as three or more nuclei grouped together, with the distance between adjacent nuclei less than their diameter) (Fig. 2B,C). Statistical data showed that over 99% of Syne-1-/- muscle fibers contained more than three nuclear clusters (Fig. 2D). These results indicate that myonuclei lacking anchorage float freely in Syne-1-/- mice, and that Syne-1 is essential to properly anchor non-synaptic nuclei and to create the space between them.
Anchorage of synapse-associated nuclei is abolished in Syne-1-/- mice
Syne-1 has been shown to be concentrated at synaptic nuclei
(Apel et al., 2000). In
previous analysis using transgenic mice expressing the C-terminus of Syne-1,
the number of synaptic nuclei was drastically reduced while the number of
nuclei peripheral to synapse was almost equally increased so that the total
number of nuclei associated with a synapse was essentially unchanged
(Grady et al., 2005). Given the
caveats of the dominant-negative effect of this transgene, as mentioned
earlier, it is essential to examine Syne-1-/- mice to
understand the role of Syne-1 in synaptic-nuclei positioning.
We stained muscle fibers with BTX (marking the AChRs) and labeled myonuclei
simultaneously with DAPI and anti-SUN2. In this study, we followed the methods
of Grady et al. (Grady et al.,
2005) to define a nucleus to be synaptic or perisynaptic: a
nucleus was defined as synaptic when at least 25% of the DAPI and SUN2 signal
overlapped with the BTX-positive site, and a perisynaptic nucleus was counted
if the DAPI and SUN2 signal did not overlap with the BTX-positive site but was
less than half its diameter from the edge of a site. Statistical data based on
DAPI staining demonstrated that the number of synaptic nuclei was
significantly reduced (data not shown). However, considering the potential
noise of non-muscle cell nuclei stained by DAPI, we further analyzed nuclear
positioning based on SUN2 signals.
In wild-type and Syne-1+/- mice, there were, on
average, approximately 4-5 nuclei clustered under each NMJ (n=140)
(Fig. 3A'''). By contrast,
no nuclei were seen under the NMJ in Syne-1-/- mice
(n=210, P<0.0001; Fig.
3B''',C''',D). This defect is significantly more severe
than the previous observation using MCK-Syne-1 KASH transgenic mice,
where 1.5 nuclei on average were still observed under the NMJ
(Grady et al., 2005).
Surprisingly, there was an average of only 0.8 perisynaptic nuclei in
Syne-1-/- mice, compared with zero in their littermate
controls (P<0.0001; Fig.
3). In MCK-Syne-1 KASH transgenic mice, this average was
greater than 2.5 nuclei (Grady et al.,
2005). Therefore, the total number of synapse-associated nuclei
(synaptic nuclei plus perisynaptic nuclei) was dramatically reduced in
Syne-1-/- mice [0.8 in Syne-1-/- vs
4.7 in wild type (P<0.0001), compared with 5.0 in Syne-1
KASH transgenic mice]. Furthermore, the anchorage defects of myonuclei in
Syne-1-/- mice were repeatedly observed in different
pieces of skeletal muscle, including in the diaphragm, tibialis anterior and
triangularis sterni. These observations indicate that Syne-1 plays a
crucial role in the positioning of synaptic nuclei in skeletal muscle.
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; Starr and Han,
2002; Zhang et al.,
2001; Zhen et al.,
2002). Using the same methods described above, we found that both
synaptic nuclei and non-synaptic nuclei were properly positioned in
Syne-2-/- mice (Fig.
4B and data not shown). Thus, Syne-2 alone is not essential for
the myonuclear positioning process.
To examine a potential overlapping function of Syne-2 with that of Syne-1
in the anchoring of myonuclei, we generated transgenic mice carrying the KASH
fragment of Syne-2 driven by the MCK promoter
(Jaynes et al., 1988). In all
five lines that we obtained, the transgenic protein was localized to the
nuclear envelope in skeletal muscle, whereas endogenous Syne-1 at the nuclear
envelope was significantly decreased in two lines where the transgene was
highly expressed (Fig. 4D and
data not shown). Additionally, in those two lines, nuclei carrying transgenic
Syne-2 rarely stayed under the NMJ, and synaptic nuclei were expelled from
under the NMJ to the peripheral region
(Fig. 4F'). Thus, the
Syne-2 fragment that contained the KASH domain displayed a similar
dominant-negative effect to that of Syne-1
(Grady et al., 2005). Those
results indicate that Syne-1 and Syne-2 might share the same docking sites on
the nuclear envelope and that Syne-2 could play a regulatory role in the
anchoring of myonuclei.
Syne-1 and Syne-2 KASH-domain double-knockout mice fail to breathe and die shortly after birth
The conserved protein structure between Syne-1 and Syne 2, their
overlapping expression patterns and the above results in Syne-2 transgenic
mice suggest that Syne-1 and Syne-2 may have redundant
functions. Thus, we examined the consequences of deleting both KASH domains.
Syne-1- and Syne-2-homozygous mutants were crossed to
generate double-heterozygous mice, which were then crossed to produce
Syne-1+/-; Syne-2-/- and
Syne-1-/-; Syne-2+/- mice. The Syne
double-heterozygous, Syne-1+/-; Syne-2-/- and
Syne-1-/-; Syne-2+/- mice were viable and
fertile. Syne-1+/-; Syne-2-/- and
Syne-1-/-; Syne-2+/- mice were then
inter-crossed to obtain Syne-1-/- and Syne-2-/-
double-homozygous-knockout (referred to hereafter as Syne DKO) mice.
Although Syne-1+/-; Syne-2+/-, Syne-1-/-;
Syne-2+/- and Syne-1+/-;
Syne-2-/- mice were born at percentages consistent with the
expected Mendelian ratio, we could not obtain viable Syne DKO mice
when genotyping at or after postnatal day 7.
Careful analysis indicated that Syne DKO mice were born alive but died within 20 minutes and were soon cannibalized by the mothers. While these double-homozygous pups had a similar body weight and anatomy to their littermates, they were cyanotic at birth and unable to breathe even though they were able to open the mouth (Fig. 5B and data not shown). Their hearts beat for a few minutes prior to death. In addition, the Syne DKO babies could move their legs in response to a painful stimulus, but failed to move their ribcages. Postmortem histological analysis of the lungs demonstrated that the alveoli air sacs of these mice were not expanded (Fig. 5F), although their skeletal muscle displayed grossly normal architecture (Fig. 5C,D).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However,
no severe physiological defects were observed to accompany the cellular
disorder, and the anchorage of non-synaptic nuclei appeared normal in these
transgenic animals. Because the dominant-negative effect of the transgene
reduced but did not eliminate Syne-1 proteins from the nuclear envelope, it
was thus not clear to what extent the Syne-1 protein is required for nuclear
anchorage in muscle cells. Additionally, because both Syne-1 and Syne-2 are
expressed in skeletal muscle cells and both could interact with the same SUN
proteins to mediate localization to the nuclear envelope, it is not clear
whether the dominant-negative effect of overexpressing the KASH domain of
Syne-1 is specific to the Syne-1 gene. Our result that the expression
of the KASH fragment of Syne-2 displayed a similar dominant-negative effect to
that of Syne-1 (Fig. 4) further
raised this concern and indicates the importance of carrying out analysis
using loss-of-function alleles.
Syne-1-/- mice displayed an almost complete loss of
synaptic nuclei, which was far more severe than that described in the previous
transgenic study (Grady et al.,
2005). The difference may be caused by the low level of nuclear
envelope-associated endogenous Syne-1 in the transgenic mice, which was
sufficient to trap the migrating myonuclei at the postsynaptic region, but was
not sufficient to stably anchor them when the muscle underwent violent
contractions. In Syne-1-/- mice, both the trapping and the
anchorage processes were disrupted.
Strikingly, we found that Syne-1 is crucial for the positioning of
not only synaptic nuclei, but also of non-synaptic nuclei. In addition,
approximately 2% of the muscle cells of Syne-1-/- mice
exhibited centralized nuclei, compared with less than 0.5% in the control
group (see Fig. S1 in the supplementary material). The severely disrupted
organization of non-synaptic nuclei could impair normal nuclear-cytoplasm
transportation, as well as other interactions, in the large syncytial muscle
cells. This could then cause the weakening of the normal functions of skeletal
muscle. This important phenotype was not observed in the previous transgenic
mice expressing the KASH fragment of Syne-1
(Grady et al., 2005), again
indicating that there was still a considerable level of Syne-1 on the nuclear
envelope that was sufficient to anchor the myonuclei in the transgenic
mice.
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C. elegans SUN-domain-containing proteins (e.g. UNC-84, SUN-1)
have been shown to play important roles in nuclear positioning by recruiting
KASH-domain proteins to the nuclear envelope
(Malone et al., 2003;
Starr et al., 2001). In
tissue-culture cells, Syne-1 and Syne-2 have also been shown to localize to
the nuclear envelope in a SUN-domain-protein-dependent manner
(Crisp et al., 2006;
Padmakumar et al., 2005) (X.D.
and X.Z., unpublished data). Thus, mouse SUN1 and SUN2 are likely to be the
partners of Syne proteins and probably play roles in myonuclear anchorage.
Synaptic nuclei and NMJ development
Synaptic nuclei have long been proposed to be transcriptionally specialized
to maintain the postsynaptic components (see Introduction), but the importance
of those nuclei has never been studied in mutant mice that lacked them.
Syne-1-/- and Syne DKO mice provide an effective
model system to study this issue. Surprisingly, neither presynaptic
(synaptophysin) nor postsynaptic (AChR, rapsyn, MuSK and utrophin) components
were found to be obviously depleted from the NMJs of Syne DKO mutants
(see Fig. S2 in the supplementary material). In addition, the termini of
phrenic nerves, which were labeled with a mixture of anti-synaptophysin and
anti-neurofilament, co-localized well with AChR patches in E18.5 Syne
DKO embryos (data not shown). These results suggest that the Agrin-MuSK-Rapsn
pathway, which is crucial for the development of the NMJ
(DeChiara et al., 1996;
Gautam et al., 1995), is not
obviously affected in Syne-1-/- or Syne DKO
mice.
However, given that NMJ-specific genes were not completely depleted, the loss of synaptic nuclei might reduce the expression level of those genes and weaken the normal functions of the NMJ. Consistent with this notion, we observed that the phenic nerves displayed longer branches in both Syne-1-/- and Syne DKO mice.
Therefore, synaptic nuclei may play important roles in selecting or maintaining the innervation sites by strengthening the communication between nerve and muscle at the newly formed muscle-nerve contacts.
The cause of neonatal lethality of Syne DKO mice
Because we failed to identify major differences in the muscle morphology,
NMJ structure and motor-nerve branching between the Syne-1-/-;
Syne-2+/- and the Syne DKO embryos, we carried out an
electrophysiological experiment and examined the endplate potential (EPP) to
find out whether the neuromuscular transmission was blocked in Syne
DKO mutants. However, the paired-pulse ratio and the decay of the evoked EPP
in E18.5 Syne DKO diaphragms showed no significant difference from
that of the Syne-1-/-; Syne-2+/- embryos (see
Fig. S3 in the supplementary material). This result suggests that the NMJ in
Syne DKO mutants may remain functional in transmitting synaptic
potentials from a motor neuron terminal to its corresponding muscle cell. The
neonatal Syne DKO mutants may die of downstream defects inside the
skeletal muscle cells, such as the intracellular-Ca2+ mobilization
and the excitation-contraction coupling of the muscle.
Thus, the cause of the respiration failure and lethality associated with Syne DKO mice remains obscure. Although the observed neonatal lethality may be due to an unknown cellular function associated with the two Syne proteins, such as defects during the development of the central nervous system, it is still conceivable that the additive effects that knocking out both genes has on disrupting nuclear anchorage is the fundamental cause of the fatality.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/901/DC1
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ACKNOWLEDGMENTS |
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