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First published online 31 January 2007
doi: 10.1242/dev.02783

Development 134, 901-908 (2007)
Published by The Company of Biologists 2007
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Syne-1 and Syne-2 play crucial roles in myonuclear anchorage and motor neuron innervation

Xiaochang Zhang1, Rener Xu1,*, Binggen Zhu1, Xiujuan Yang2, Xu Ding1, Shumin Duan2, Tian Xu1,3, Yuan Zhuang1,4 and Min Han1,5

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.


Figure 1
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  		Fig. 1 . The generation of Syne-1 and Syne-2 KASH-domain-deletion mice. (A,B) Schematic representation of the knockout strategies for Syne-1 and Syne-2. Exons are labeled and coding regions are indicated by black boxes. In the targeting vector, the last exon of Syne-1 (the last two exons of Syne-2) was replaced by a neomycin-resistance expression cassette (neo). A HSV-TK cassette was linked to the 5' end (3' end for Syne-2) for negative selection. Restriction enzyme sites: A, AvrII; B, BamHI; E, EcoRI; H, HindIII. (C,D) Southern-blot analyses of genomic DNA from wild-type (+/+), heterozygous (+/-) and homozygous-knockout (-/-) mice. Probes used are indicated in Fig. 1A,B. (C) For Syne-1 analysis, genomic DNA samples were digested by BamHI, which yielded 3.8 kb (wild-type allele) and 5.2 kb (mutant allele) bands (notice that a 4.1 kb band caused by BamHI digestion is visible in all lanes). (D) For Syne-2, SpeI-digested genomic DNA yielded 5.5 kb (wild-type allele) and 2.6 kb (mutant allele) bands. (E-H) Frozen sections of skeletal muscle were stained with anti-Syne-1 (green in E and F) or anti-Syne-2 (green in G and H), and DAPI (blue in all panels). Syne-1 and Syne-2 signals are visible on the nuclear envelope of samples from the control, but not the homozygous-knockout, mice. Scale bar, 25 µm in F for E,F; 10 µm in H for G,H.

 

Figure 2
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  		Fig. 2 . Non-synaptic nuclei are disorganized in Syne-1-/- mice. (A-C) A single muscle fiber was teased from the tibialis anterior and simultaneously stained with DAPI (blue) and anti-SUN2 (green). Myonuclei were found to be distributed evenly in Syne-1+/- (A), but formed clusters (B) and arrays (C) in Syne-1-/- mice. (D) Statistical data shows that more than 99% of fibers in Syne-1-/- mice formed three or more nuclear clusters outside NMJs (n>110 for each group). Scale bar: 25 µm.

 

Figure 3
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  		Fig. 3 . Anchorage of synaptic nuclei is abolished in Syne-1-/- mice. (A-A''') Representative Syne-1+/- muscle fiber with four nuclei (synaptic nuclei) clustered under the NMJ. (B-C''') No nuclei were observed under the NMJ of Syne-1-/- mice. (D,E) Statistical analyses of synaptic nuclei based on anti-SUN2 signals. Synaptic nuclei disappeared in Syne-1-/- mice (4.7 in Syne-1+/+ and Syne-1+/- vs 0.0 in Syne-1-/-, n=140 for Syne-1+/? and 210 for Syne-1-/-, P<0.0001); the number of perisynaptic nuclei was slightly increased in Syne-1-/- mice (0.0 in control vs 0.8 in KO, P<0.0001). Blue, DAPI; Green, anti-SUN2-labeled myonuclei; Red, BTX. Scale bar: 25 µm.

 

Figure 4
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  		Fig. 4 . Myonuclei positioning is not affected in Syne-2-/- mice, but an MCK-driven Syne-2 KASH transgenic protein displays a dominant-negative effect. (A,B) Anchorage of synaptic nuclei in Syne-2-/- mice (B) was similar to wild-type mice (A). Green, anti-SUN2 labeled myonuclei; red, BTX. (C,D) An MCK-promoter-driven C-terminal fragment of Syne-2 containing the KASH domain was localized to the nuclear envelope in skeletal muscle cells (D). Blue, DAPI; green, anti-myc. (E-F') Overexpressed Syne-2 KASH fragment expelled synaptic nuclei from under the NMJ to the peripheral region (F,F'), compared with wild-type (E,E'). Noticeably, myonuclei expressing the transgene (green) rarely stayed under synapses (F'). Scale bars: 25 µm in B for A,B; 25 µm in C for C,D; 25 µm in F for E-F'.

 

Figure 5
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  		Fig. 5 . Syne-1 and Syne-2 double-knockout mice die soon after birth. (A,B) Newborn Syne DKO mice (B) appeared cyanotic at birth compared to their double-heterozygous littermates (A). (C,D) Longitudinal sections of E18.5 diaphragms. Syne DKO embryo displayed grossly normal muscle anatomy (D) compared to Syne-1+/-; Syne-2+/- (C). (E,F) Postmortem histological analysis of the lung showed that the alveoli air sacs of DKO mice were not expanded (F), indicating the failure of breathing. A Syne-1+/-; Syne-2+/- littermate was used as the control (E). Scale bars: 25 µm in D for C,D; 100 µm in F for E,F.

 

Figure 6
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  		Fig. 6 . Nuclear-anchorage defects in muscle of Syne DKO mice. (A-D) Representative NMJs from E18.5 triangularis sterni of the four different Syne-knockout genotypes. Sy1 and Sy2 represent Syne-1 and Syne-2, respectively. Blue, DAPI; red, BTX. Notice that one synaptic nucleus (blue) stayed under the NMJ (red) in A and C, but not in B or D. (E) Statistical data showed that the Syne-1-/-; Syne-2+/- and Syne DKO embryos displayed a significant loss of synaptic nuclei compared with embryos of the other two genotypes. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7 . Phrenic nerves display longer branches in Syne-1-/- and Syne DKO mutants. (A-D) Whole-mount diaphragms of E18.5 embryos were stained with anti-neurofilament and anti-synaptophysin (green). The right hemi-diaphragm of each genotype is shown. Longer branches are obvious in Syne-1-/-; Syne-2+/- and Syne-1-/-; Syne-2-/- samples. (A'-D') Enlarged views of asterisk-indicated regions in A-D, showing the elongated phrenic nerve branches (green) and the broader endplate bands (red) in Syne-1-/-; Syne-2+/- and Syne-1-/-; Syne-2-/- diaphragms (C',D'). Scale bars: 500 µm in D for A-D; 50 µm in D' for A'-D'.

 




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