Slit2 as a β-catenin/Ctnnb1-dependent retrograde signal for presynaptic differentiation
The neuromuscular junction ( NMJ ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells to control muscle contraction . It exhibits a high degree of subcellular specialization characteristic of chemical synapses and has served as a model to study synaptogenesis ( Sanes and Lichtman , 2001; Wu et al . , 2010; Barik et al . , 2014b ) . Abnormal NMJ formation or function causes neurological disorders including congenital myasthenic syndrome and myasthenia gravis . NMJ formation requires reciprocal interactions between nerve terminals and muscle cells . In anterograde signaling , nerve-derived agrin directs post-synaptic differentiation by interacting with muscle transmembrane receptors LRP4 and MuSK , which in turn activate downstream signaling events leading to AChR concentration at the NMJ ( McMahan , 1990; DeChiara et al . , 1996; Glass et al . , 1996; Weatherbee et al . , 2006; Kim et al . , 2008; Zhang et al . , 2008 ) . However , much less is known regarding retrograde mechanisms that control motor neuron survival and presynaptic differentiation . Wnt signaling has been implicated in NMJ formation in Caenorhabditis elegans , Drosophila as well as vertebrates ( Korkut and Budnik , 2009; Zhang et al . , 2009; Barik et al . , 2014b ) . β-Catenin ( Ctnnb1 ) is a central component of the canonical Wnt signaling pathway , regulating transcription by associating with TCF/LEF transcription factors ( MacDonald et al . , 2009 ) . It also regulates adhesion-dependent signaling by binding to cadherins and α-catenin ( Nelson and Nusse , 2004 ) . Intriguingly , when Ctnnb1 is mutated in muscle fibers , mutant mice die neonatally , with profound presynaptic deficits such as mislocation of phrenic nerve primary branches , reduced synaptic vesicles , and impaired neuromuscular transmission ( Li et al . , 2008 ) , suggesting that Ctnnb1 in muscle cells is necessary for presynaptic differentiation . In support of this notion are recent reports that expression of stable Ctnnb1 in muscle cells also impairs presynaptic differentiation in mutant mice ( Liu et al . , 2012; Wu et al . , 2012a ) . These observations suggest that Ctnnb1 in muscle is critical for a retrograde pathway to direct nerve terminal development . However , the underlying mechanism remains unclear . The interaction of cadherins of pre- and post-synaptic membranes has been shown to be important for synapse formation ( Bamji et al . , 2003; Bozdagi et al . , 2004; Prakash et al . , 2005 ) and synaptic plasticity ( Murase et al . , 2002; Schuman and Murase , 2003; Nuriya and Huganir , 2006 ) . NMDAR stimulation accumulates Ctnnb1 in spines , which in turn regulates induced endocytosis of N-cadherins ( Tai et al . , 2007 ) . These observations raise questions whether the muscle Ctnnb1 regulates presynaptic differentiation via cell adhesion-dependent signaling and/or gene-expression . In this paper , we determined which function of Ctnnb1 is required for NMJ formation by characterizing transgenic mice expressing wild-type or Ctnnb1 mutants that were impaired in transcriptional regulation or cell-adhesion signaling . Rescue experiments indicated a necessary role for the transcription activity of muscle Ctnnb1 in presynaptic differentiation . Our exploration of targets of Ctnnb1 as potential muscle-derived retrograde factors led to the identification of Slit2 , an environmental cue that repels or collapses neuronal axons ( Brose et al . , 1999; Kidd et al . , 1999 ) . Slit2 belongs to a family of large ECM ( extracellular matrix ) glycoproteins known to be chemorepellent for olfactory , motor , hippocampal , and retinal axons ( Nguyen Ba-Charvet et al . , 1999; Erskine et al . , 2000; Niclou et al . , 2000; Ringstedt et al . , 2000 ) . However , Slit2 was also shown to stimulate the formation of axon collateral branches by dorsal root ganglia neurons ( Wang et al . , 1999 ) and positively regulate motor axon fasciculation ( Jaworski and Tessier-Lavigne , 2012 ) . Slit2 was able to induce clusters of synaptophysin in cultured neurons , suggesting a synaptogenic function . Expression of Slit2 specifically in muscle fibers was able to rescue NMJ deficits in Ctnnb1-mutant mice . These observations demonstrate that Ctnnb1 regulates presynaptic differentiation by a transcription-dependent mechanism and identify Slit2 as a novel retrograde factor in NMJ formation .
The N-terminal region of Ctnnb1 interacts with α-catenin , critical for cell adhesion . The key amino acid residues in Ctnnb1 for interaction with α-catenin have been mapped to Thr-120 and Val-122 ( Aberle et al . , 1996a , 1996b ) . Mutation of these two residues to alanines prevents Ctnnb1 from binding to α-catenin ( Xu et al . , 2000 ) . However , the mutation has no effect on binding to TCF/Lef1 , and thus , does not alter the transcription by Ctnnb1 and TCF/Lef1 ( Xu et al . , 2000 ) . On the other hand , the transcriptional regulation requires the transactivation domain ( TAD ) ( Molenaar et al . , 1996; van de Wetering et al . , 1997; Vleminckx et al . , 1999 ) . To determine which function of Ctnnb1 is necessary , we generated transgenic mice: Ctnnb1TV-AA ( LSL-Ctnnb1TV-AA ) to impair the cell-adhesion function and TAD-deleted Ctnnb1 ( LSL-Ctnnb1ΔTAD ) to eliminate the transcriptional regulation , as well as wild-type LSL-Ctnnb1 . Each transgene was placed downstream of a loxP-flanked transcriptional STOP ( LSL ) and contained the lacZ/neomycin fusion gene and triple repeats of the SV40 polyadenylation signal ( Zinyk et al . , 1998 ) . Expression of the transgenes , which were tagged by an HA epitope in the C-terminus , was under the control of the CAG promoter ( the CMV enhancer/chicken β-actin promoter ) and depended on expression of a Cre recombinase ( Zinyk et al . , 1998 ) ( Figure 1—figure supplement 1A , B ) . In HEK293 cells , co-expression of GFP-Cre , which floxed out the stop element , led to expression of Ctnnb1 , Ctnnb1TV-AA , and Ctnnb1ΔTAD that could be detected by both anti-HA and anti-Ctnnb1 antibodies ( Figure 1—figure supplement 1C ) . To determine whether Ctnnb1 and mutants were expressed in skeletal muscles , the transgenic mice were crossed with ACTA1-Cre mice that express the Cre recombinase specifically in skeletal muscles under the control of the promoter of the human skeletal alpha-actin ( HSA or ACTA1 ) gene ( Miniou et al . , 1999; Li et al . , 2008; Wu et al . , 2012a , 2012b ) . As shown in Figure 1—figure supplement 1C , D , HA-tagged Ctnnb1 and mutants were expressed in muscles of ACTA1-Cre::LSL-Ctnnb1 , but not in muscles of ACTA1-Cre or mice that carried only the transgenes . Moreover , the transgene expression was muscle-specific and not detectable in other tissues including spinal cord ( Figure 1—figure supplement 1D , E ) ( data not shown ) . ACTA1-Cre::LSL-Ctnnb1 , ACTA1-Cre::LSL-Ctnnb1TV-AA , and ACTA1-Cre::LSL-Ctnnb1∆TAD mice were viable and fertile and exhibited normal NMJ morphology , compared to control mice ( ACTA1-Cre or Ctnnb1fl/fl ) ( Figure 1—figure supplement 1F ) ( data not shown ) . No difference was observed in location of primary branches of the phrenic nerves , secondary branches , and location and size of AChR clusters at postnatal day 0 ( P0 ) between controls and mutants ( Figure 1—figure supplement 1F ) ( data not shown ) . We bred the LSL-transgenic mice with ACTA1-Cre::Ctnnb1fl/+ mice ( ACTA1-Ctnnb1fl/+ ) to determine whether and which transgene was able to rescue NMJ deficits by Ctnnb1 mutation . Notice that both the expression of wildtype or mutant Ctnnb1 and the deletion of endogenous Ctnnb1 depend on the ACTA1 promoter . In control mice ( Ctnnb1fl/fl ) , primary branches of the phrenic nerve were located in the middle of muscle fibers . The secondary branches were numerous and short , many of which end with AChR clusters that were confined to the central region of muscle fibers ( Lin et al . , 2000; Li et al . , 2008 ) ( Figure 1A ) . However , in ACTA1-Ctnnb1−/− ( i . e . , CATA1::Cre;Catnnb1f/f ) mutants , the primary branches were mislocated to the tendon region close to the central cavity , the secondary branches were fewer and longer , and the region where AChR cluster distributed became wider ( Figure 1 ) . As expected , expression of wild-type Ctnnb1 was able to rescue these phenotypes ( Figure 1 ) . Interestingly , these phenotypes were also completely rescued by Ctnnb1TV-AA since no difference was detectable in primary branch location , secondary branch number and length , and endplate band width between ACTA1-Ctnnb1−/−::LSL-Ctnnb1ΔTAD mice and control or ACTA1-Ctnnb1−/−::LSL-Ctnnb1 mice , indicating that the cell-adhesion function of Ctnnb1 is not necessary for presynaptic differentiation . In contrast , however , the TAD deletion mutant was unable to rescue these phenotypes , suggesting a requirement of the transcription function ( Figure 1 ) . 10 . 7554/eLife . 07266 . 003Figure 1 . Requirement of the TAD domain to rescue presynaptic deficits in ACTA1-Ctnnb1−/− deficits . ( A ) Diaphragms of P0 mice of indicated genotypes were stained whole mount with rhodamine-conjugated α-BTX ( R-BTX ) to label AChR clusters and anti-NF/synaptophysin antibodies ( green ) to label axons and nerve terminals . Shown were left ventral areas . Red arrowhead , primary branches; blue arrowheads , secondary branches . M , medial; L , lateral . Diagrams summarizing morphological deficits . ( B–E ) Quantitative analysis of data in A . Data were shown as mean ± SEM; ** , p < 0 . 01; *** , p < 0 . 001; One-way ANOVA; n = 4; bar , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 00310 . 7554/eLife . 07266 . 004Figure 1—figure supplement 1 . Generation and identification of LSL-Ctnnb1 , LSL-Ctnnb1TV-AA , LSL-Ctnnb1ΔTAD transgenic mice . ( A ) The wild-type , TV-AA , and ΔTAD Ctnnb1 were tagged at the C-terminus by HA-tag and were subcloned into the pCCALL2 vector . ( B ) Breeding scheme to generate rescue genotypes . ( C ) Western blot showing Cre-dependent expression of wild-type , TV-AA , and ΔTAD Ctnnb1 transgenes in HEK293 cells . ( D ) Western blot showing ACTA1-Cre-dependent expression of Ctnnb1 transgenes in vivo . ( E ) Western blot showing HA-tagged Ctnnb1TV-AA expressed only in the muscles of ACTA1-Ctnnb1−/−::LSL-Ctnnb1TV-AA , with concomitant deletion of endogenous Ctnnb1 alleles were knocked out . ( F ) Diaphragms of P0 mice of indicated genotypes were stained whole mount with R-BTX to label AChR clusters ( red ) and anti-NF/synaptophysin antibodies ( green ) to label axons and nerve terminals . Shown were left ventral areas . Red arrowhead , primary branches; blue arrowheads , secondary branches . M , medial; L , lateral; bar = 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 004 We have shown that muscle-specific ablation of Ctnnb1 disrupts presynaptic structure and function ( Li et al . , 2008; Wu et al . , 2012a ) . To determine whether the presynaptic deficits could be rescued , we performed electron microscopic analysis . In control mice , axon terminals were filled with synaptic vesicles , some of which were docked on electron-dense active zones ( Figure 2 ) . The vesicle density in control terminals was 3 . 6 ± 0 . 30 vesicles/0 . 04 μm2 . In contrast , the density was reduced to 1 . 40 ± 0 . 37 vesicles/0 . 04 μm2 in ACTA1-Ctnnb1−/− terminals ( p < 0 . 001 ) , in agreement with previous report ( Liu et al . , 2012; Wu et al . , 2012a ) . As shown in Figure 2 , the reduction in vesicle density was diminished by expressing Ctnnb1 or Ctnnb1TV-AA , with 3 . 6 ± 0 . 33 and 3 . 99 ± 0 . 33 vesicle/0 . 04 mm2 in ACTA1-Ctnnb1−/−::LSL-Ctnnb1 mice and ACTA1-Ctnnb1−/−::LSL-Ctnnb1TV-AA mice , respectively ( p < 0 . 001 in comparison with ACTA1-Ctnnb1−/− , n = 17 ) ( Figure 2A , B ) . These results indicated that presynaptic deficits could be rescued by wild-type Ctnnb1 or Ctnnb1TV-AA , suggesting a dispensable role of the cell-adhesion function of Ctnnb1 . In contrast , synaptic vesicle density in ACTA1-Ctnnb1−/−::LSL-Ctnnb1∆TAD remained low , at 1 . 33 ± 0 . 50 vesicles/0 . 04 μm2 , which was not different from ACTA1-Ctnnb1−/− mice ( p > 0 . 05 in comparison with control; n = 17 ) ( Figure 2A , B ) , demonstrating inability of the ΔTAD mutant to rescue and suggesting an important role of the transcription regulation in the retrograde pathway . Similarly , active zones were fewer in ACTA1-Ctnnb1−/− mice and ACTA1-Ctnnb1−/−::LSL-Ctnnb1∆TAD mice , compared with control mice , ACTA1-Ctnnb1−/−::LSL-Ctnnb1 mice , and ACTA1-Ctnnb1−/−::LSL-Ctnnb1TV-AA mice ( Figure 2A , C ) . As reported previously ( Wu et al . , 2012a ) , Ctnnb1 mutation in muscles did not alter the number of terminals per NMJ and the width of synaptic clefts . Expression of Ctnnb1 wild type or mutants did not change these parameters ( Figure 2A , D , E ) . These results are in agreement with the notion that muscle Ctnnb1 mainly regulates presynaptic differentiation . 10 . 7554/eLife . 07266 . 005Figure 2 . The TAD is necessary for muscle Ctnnb1 to regulate presynaptic development . Diaphragms of P0 mice were subjected to electron microscopic analysis . ( A ) Representative electron micrographic images of indicated genotypes . Asterisks , active zones; SBL , synaptic basal lamina; N , nerve terminals; M , muscle fibers; SC , Schwann cells; SV , synaptic vesicle . ( B–E ) Quantitative analysis of data in A . Data were shown as mean ± SEM; ** , p < 0 . 01; *** , p < 0 . 001 , One-way ANOVA , n = 10 . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 00510 . 7554/eLife . 07266 . 006Figure 2—figure supplement 1 . Synaptic nuclear localization of Ctnnb1 and Axin2-nlacZ in the NMJ region . Muscle fibers were stained whole mount with anti-Ctnnb1 ( A , green ) or for β-galactosidase ( B , red ) , together with DAPI to label nuclei and R-BTX to label AChR . bar = 10 μm . NMJ , neuromuscular junction . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 006 In addition to morphological studies , we measured miniature end-plate potentials ( mEPPs ) , events that are generated by spontaneous vesicle release . There was about 50% reduction in mEPP frequency in ACTA1-Ctnnb1−/− mice , compared with control mice ( Li et al . , 2008 ) ( Figure 3A , B ) ( 1 . 04 ± 0 . 04 Hz for control; 0 . 45 ± 0 . 04 Hz for ACTA1-Ctnnb1−/− , p < 0 . 001; n = 4 ) , but no change in mEPP amplitude ( Figure 3A , C ) . These results were in agreement with morphological presynaptic deficits ( Figures 1 , 2 ) . The frequency reduction phenotype was rescued by both wild-type Ctnnb1 and the TV-AA mutant ( 0 . 95 ± 0 . 09 Hz in ACTA1-Ctnnb1−/−::LSL-Ctnnb1 and 1 . 06 ± 0 . 04 Hz in ACTA1-Ctnnb1−/−::LSL-Ctnnb1TV-AA , p > 0 . 05 in comparison with control; n = 4 ) ( Figure 3A , B ) . In contrast , mEPP frequency remained depressed in ACTA1-Ctnnb1−/−::LSL-Ctnnb1ΔTAD mice ( Figure 3A , B ) . Together , these results of both morphological and functional studies indicate the requirement of the Ctnnb1's TAD in motor nerve terminal differentiation and suggest that Ctnnb1-regulated transcription is necessary for motor nerve terminal differentiation . 10 . 7554/eLife . 07266 . 007Figure 3 . Rescue of neurotransmission deficits depends on Ctnnb1 TAD domain . miniature end-plate potentials ( mEPPs ) were recorded in P0 diaphragms of indicated genotypes . ( A ) Representative mEPP traces . ( B–C ) Quantitative analysis of mEPP amplitude ( B ) and frequency ( C ) . Data were shown as mean ± SEM; *** , p < 0 . 001; One-way ANOVA; n = 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 007 To determine whether Ctnnb1 is locally activated , we performed the following two experiments ( Figure 2—figure supplement 1 ) . First , whole-mount staining of muscle fibers with Ctnnb1 antibody indicates that Ctnnb1 in synaptic nuclei appeared to be higher than that in extra-synaptic regions ( Figure 2—figure supplement 1A ) . Second , we characterized Axin2-nlacZ mice , where the nuclear-localized β-galactosidase ( nlacZ ) DNA was inserted in the Axin2 gene , a target of Wnt/Ctnnb1 signaling . Expression of β-galactosidase in Axin2-nlacZ mice is controlled , which has been used as a reporter of Wnt or Ctnnb1 activity ( Lustig et al . , 2002 ) . As shown in Figure 2—figure supplement 1B , β-galactosidase was detectable in synaptic nuclei , not in extra-synaptic nuclei . These results are in agreement with the notion of active Wnt/Ctnnb1 signaling in synaptic nuclei at the NMJ and provide additional support to the model . The necessity of the TAD in muscle Ctnnb1 regulation of presynaptic terminals suggests that genes whose expression is controlled by Ctnnb1 may serve as retrograde factors . To identify such a factor , we performed quantitative real-time PCR ( qRT-PCR ) to screen for genes that are down-regulated in ACTA1-Ctnnb1−/− muscles , compared with Ctnnb1fl/fl control . We focused on secretable and transmembrane proteins in particular muscle-derived extracellular matrix proteins , trophic factors , morphogens , and environmental cues ( Figure 4B–E ) . C-Myc and Axin2 , whose expression is regulated by Ctnnb1 ( He et al . , 1998; Yan et al . , 2001 ) , were used a positive control . Their mRNAs were lower in ACTA1-Ctnnb1−/− muscles than control , indicating the validity of the method ( Figure 4A ) . In a panel of more than 70 genes , 9 were reduced in expression in ACTA1-Ctnnb1−/− , compared to control Ctnnb1fl/fl mice ( Figure 4 ) . Of particular interest is Slit2 , a repulsive axon guidance cue of the Slit family ( Brose et al . , 1999; Kidd et al . , 1999 ) . Slit2 as well as Slit1 regulate axon collateral formation ( Nguyen Ba-Charvet et al . , 1999 ) . Interestingly , mutation of Slit2 , but not Slit1 or Slit3 , disrupts motoneuron fasciculation and primary nerve location ( Jaworski and Tessier-Lavigne , 2012 ) , akin to NMJ morphological deficits of ACTA1-Ctnnb1−/− mice . Of the three members of the Slit family , only Slit2 was down-regulated in ACTA1-Ctnnb1−/− muscles ( Figure 4E ) . The reduction was also confirmed by Western blot analysis of ACTA1-Ctnnb1−/− muscle homogenates ( Figure 5A , B ) . 10 . 7554/eLife . 07266 . 008Figure 4 . Slit2 was reduced in ACTA1-Ctnnb1−/− muscle . RNA was purified from P0 muscles of Ctnnb1fl/fl ( black ) and ACTA1-Ctnnb1−/− ( red ) and subjected to quantitative real-time PCR . Of 70 genes , the expression of 25 was negligible . Shown were genes in different groups: known Ctnnb1 targets ( A ) , ECM ( extracellular matrix ) proteins ( B ) , trophic factors ( C ) , morphogens ( D ) , and environmental cues ( E ) . Data are shown as mean ± SEM; * , p < 0 . 05; ** , p < 0 . 01; *** , p < 0 . 001; t-test; n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 00810 . 7554/eLife . 07266 . 009Figure 5 . Transcriptional regulation of Slit2 by Ctnnb1 . ( A ) Expression of Slit2 was reduced in ACTA1-Ctnnb1−/− muscles . ( B ) Quantitative analysis of data in A . Data are shown as mean ± SEM; ** , p < 0 . 01; t-test; n = 3 . ( C ) Slit2 expression was increased by elevating Ctnnb1 . C2C12 myoblasts were transfected with HA-Ctnnb1 . Resulting myotubes were subjected to Western blot with antibodies against Slit2 or HA . ( D ) Diagram showing putative TCF4/LEF1 binding sites in 5′-UTR . Sites were identified by ChIP-MAPPER ( Marinescu et al . , 2005 ) . Primers for CHIP analysis were indicated by arrows . ( E ) Ctnnb1 binding to the promoter of Slit2 5′UTR ( red arrowhead ) . Cross-linked DNA of C2C12 myotubes was subjected precipitation with anti-Ctnnb1 antibody . The complex was used as a template for PCR with the primers indicated in D . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 009 Muscle homogenates contain , in addition to muscle , nerve terminals , Schwann cells , and blood vessels and connective tissues where Slit2 may be expressed ( Huang et al . , 2011; Jaworski and Tessier-Lavigne , 2012 ) . To demonstrate that Slit2 is expressed in muscle cells and the expression is regulated by Ctnnb1 , we transfected C2C12 myoblasts with HA-tagged Ctnnb1 and resulting myotubes were subjected to Western blotting for endogenous Slit2 . As shown in Figure 5C , elevating Ctnnb1 level increased the expression of endogenous Slit2 in a dose-dependent manner . This result indicates that Ctnnb1 stimulates the expression of Slit2 in muscle cells . Ctnnb1 regulates gene expression by binding to the Tcf4/Lef1 transcription factors ( Behrens et al . , 1996; van de Wetering et al . , 1997 ) . We identified several putative Tcf4/Lef1-binding sites in the 5′UTR of the Slit2 gene using ChIP-MAPPER ( Marinescu et al . , 2005 ) . There were a Tcf4 and a Lef1 site between −1061 and −929 bases and two Tcf4 sites between −582 and −567 bases upstream from the transcription initiation site ( Figure 5D ) . To determine whether Ctnnb1 in fact binds to these sites , we performed chromatin immunoprecipitation ( ChIP ) assay using C2C12 myotubes . DNA fragments associated with Ctnnb1 were immunoprecipitated with anti-Ctnnb1 antibody and subjected to PCR using primers that flank these two regions ( Figure 5D ) . As shown in Figure 5E , a specific band was detected using the primers ( −582–567 ) , suggesting that Ctnnb1 is associated with the Tcf4 sites . In contrast , no band was detected using the primers ( −1061 and −929 ) , although the primers were able to detect a band in input , suggesting that the Tcf4 and Lef1 sites in this region may not be able to bind to Ctnnb1 . Together , these results suggest that Ctnnb1 binds to the promoter region of the Slit2 gene to promote its expression in muscle cells . Having demonstrated that Slit2 is a target of Ctnnb1-regulated transcription in muscles , we wanted to determine if overexpressing Slit2 in muscles was able to rescue presynaptic deficits in ACTA1-Ctnnb1−/− mice . It should if the hypothesis is correct . To this end , we generated a transgenic mouse , ACTA1-Slit2 , which expressed Flag-Slit2 under the control of the ACTA1 promoter ( Figure 6—figure supplement 1A , B ) . The ACTA1 promoter drives muscle-specific expression at embryonic day 9 . 5 ( E9 . 5 ) ( Crawford et al . , 2000 ) and has been useful to study NMJ assembly ( Kim and Burden , 2008; Wu et al . , 2012a , 2012b ) . The transgene Flag-Slit2 was expressed in muscles of ACTA1-Slit2 mice , but not muscles of control mice ( Figure 6—figure supplement 1B ) . Flag-Slit2 expression was muscle-specific and not detectable in other tissues including the spinal cord ( data not shown ) . Interestingly , many of presynaptic morphological deficits of ACTA1-Ctnnb1−/− mice were rescued by Slit2 , including reduced nerve terminal coverage ( as indicated by synaptophysin staining ) ( Figure 6A–C ) , reduced vesicle density ( Figure 7A , B ) , and reduced active zones ( Figure 7A , C ) . While the number of nerve terminals and synaptic cleft width in either ACTA1-Ctnnb1−/− mutant or ACTA1-Ctnnb1−/−::ACTA1-Slit2 rescue mice are not affected ( Figure 7D , E ) . These results indicate that overexpressing Slit2 in muscles was sufficient to diminish presynaptic structural deficits that are caused by muscle Ctnnb1 deficiency , in agreement with the notion that Slit2 acts downstream of Ctnnb1 . To test this hypothesis further , we characterized neuromuscular transmission of ACTA1-Ctnnb1−/−::ACTA1-Slit2 mice . Loss of Ctnnb1 in muscles resulted in reduced mEPP frequency but not amplitude ( Figure 7F–H ) ( Li et al . , 2008 ) . Remarkably , mEPP frequency in ACTA1-Ctnnb1−/−::ACTA1-Slit2 mice was higher than that of ACTA1-Ctnnb1−/− mice , suggesting again that the vesicle release deficit was rescued by muscle expression of Slit2 . The rescue effect of nerve terminal structure and function appeared to be specific because overexpression of Slit2 in muscles had no effect on mislocation of primary nerve branches , reduced number of secondary branches , and increased length of secondary branches ( Figure 6—figure supplement 2A–E ) . The effects of muscle expression of Slit2 on post-synaptic deficits were mixed . Ablation of Ctnnb1 in muscles ( in ACTA1-Ctnnb1−/− mice ) enlarges AChR clusters and increased the central band area where AChR clusters are distributed ( Li et al . , 2008 ) . Slit2 muscle expression had no effect on AChR cluster length or area of ACTA1-Ctnnb1−/− mice ( Figure 6A , D , E ) . However , the enlarged central band area where AChR clusters are distributed in ACTA1-Ctnnb1−/− mice was reduced ( Figure 6—figure supplement 2A , E ) . Together , these observations indicate muscle-specific expression of Slit2 was able to rescue presynaptic structural and functional deficits at nerve terminals . 10 . 7554/eLife . 07266 . 010Figure 6 . Slit2 overexpression increased synaptophysin staining at the NMJ . P0 diaphragms of indicated genotypes were stained whole mount with R-BTX ( red ) and antibodies against neurofilament to label axons ( green ) , and anti-synaptophysin antibody to label synaptic vesicles ( cyan ) . ( A ) Representative images . Arrows indicate NMJs with synaptophysin; arrowheads indicate NMJ with reduced synaptophysin . ( B–E ) Quantitative analysis of data in A . Data are shown as mean ± SEM; * , p < 0 . 05; ** , p < 0 . 01; *** , p < 0 . 001; n = 9; One-way ANOVA; bar = 10 μm . NMJ , neuromuscular junction . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 01010 . 7554/eLife . 07266 . 011Figure 6—figure supplement 1 . Generation of ACTA1-Slit2 transgenic mice . ( A ) Flag-Slit2 subcloned downstream of ACTA1 promoter used for the generation of ACTA1-Slit2 transgenic mice . ( B ) Breeding scheme to generate ACTA1-Slit2 mice . ( C ) Transgenic Slit2 expression was detected in the diaphragm muscles of ACTA1-Slit2 mice . ( D ) Quantitative analysis of C . Data are shown as mean ± SEM; ** , p < 0 . 01; t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 01110 . 7554/eLife . 07266 . 012Figure 6—figure supplement 2 . ACTA1-Slit2 partially rescues ACTA1-Ctnnb1−/− axon arborization defects . ( A ) Diaphragms of P0 mice of indicated genotypes were stained whole mount with R-BTX to label AChR clusters ( red ) and anti-NF/synaptophysin antibodies ( green ) to label axons and nerve terminals . Shown were left ventral areas . Red arrowhead , primary branches; blue arrowheads , secondary branches . M , medial; L , lateral; bar = 100 μm . Diagrams summarizing morphological deficits . ( B–E ) Quantitative analysis of data in A . Data were shown as mean ± SEM; * , p < 0 . 05; ** , p < 0 . 01; *** , p < 0 . 0001; One-way ANOVA; n = 4; bar , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 01210 . 7554/eLife . 07266 . 013Figure 7 . Rescue of nerve terminal deficits by ACTA1-Slit2 . ( A ) Rescue of nerve terminal morphology . Representative electron micrographic images of indicated genotypes . Asterisks , active zones; SBL , synaptic basal lamina; N , nerve terminals; M , muscle fibers; SC , Schwann cells; SVs , synaptic vesicles . ( B–E ) Quantitative analysis of data in A . ( F ) Rescue of mEPP frequency reduction . Shown were representative mEPP traces . ( G–H ) Quantitative analysis of mEPP amplitude and frequency , respectively . Data were shown as mean ± SEM; * , p < 0 . 05; ** , p < 0 . 01; *** , p < 0 . 001; One-way ANOVA; n = 10 for B–E; n = 4 for G–H . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 013 The finding that muscle expression of Slit2 is sufficient to increase vesicle numbers and active zones in Ctnnb1-deficient mice suggested to us that Slit2 may be synaptogenic . Once released from muscles , Slit2 may be concentrated at the NMJ , like muscle-expressed AChE ( Rotundo et al . , 2008; Barik et al . , 2014a ) . To test these hypotheses , we stained muscles with anti-Slit2 antibody . As shown in Figure 8A , the immunoreactivity co-localized with R-BTX and synaptophysin ( Figure 8A ) , indicating that Slit2 is enriched in synaptic basal lamina ( SBL ) . To determine whether concentrated Slit2 was able to induce nerve terminal differentiation , we expressed Slit2 in HEK293 cells ( Figure 8B ) . Purified Slit2 was immobilized on fluorescently conjugated latex microspheres ( beads ) , which were then incubated with spinal cord explant ( ventral horn ) in culture ( Figure 8C ) . The culture was stained for synaptophysin 24 hr later . As shown in Figure 8C–E , 46 . 9 ± 2 . 62% of Slit2-beads were positive for synaptophysin , whereas , in contrast , only 18 . 6 ± 1 . 47% of controls were synaptophysin-positive ( n = 10 , p < 0 . 001 ) ( Figure 8C , D ) . Moreover , the size of synaptophysin puncta was also increased with Slit2-conjugated beads , compared with controls ( Figure 8C , E ) . Together , these results suggest that immobilized Slit2 may be sufficient to induce synaptophysin clusters of cultured spinal cord explant . 10 . 7554/eLife . 07266 . 014Figure 8 . Slit2 was enriched at the NMJ and induced synaptophysin puncta of spinal cord explants . ( A ) P12 tibilalis anterior muscles stained with R-BTX ( red ) , anti-neurofilament antibody ( green ) , and anti-Slit2 antibody ( blue ) . ( B ) Detection of Flag-Slit2 in condition medium of transfected HEK293 cells . ( C ) Increased synaptophysin puncta co-localization with Flag-Slit2-conjugated beads . ( D–E ) Quantitative analysis of data in C , Data are shown as mean ± SEM; ** , p < 0 . 01; t-test; bar = 10 μm for A; bar = 500 μm for C . DOI: http://dx . doi . org/10 . 7554/eLife . 07266 . 014
Many molecules have been identified to regulate presynaptic differentiation at the NMJ . For example , muscle-derived FGF 7/10/22 and collagens IV and XIII were shown to induce synaptic vesicle clusters at nerve terminals or regulate presynaptic maturation ( Fox et al . , 2007; Latvanlehto et al . , 2010 ) . In Drosophila , wishful thinking and Glass bottom boat , a TGFβ receptor and ligand , respectively , were shown to be important for presynaptic T-bar formation ( Marques et al . , 2000; McCabe et al . , 2003 , 2004 ) . GDNF ( glial cell derived neurotrophic factor ) , known to affect motoneuron survival in vitro , was shown to affect pre- and post-synaptic neurotransmission in frog neuron-muscle co-cultures ( Oppenheim et al . , 1995; Keller-Peck et al . , 2001; Wang et al . , 2002 ) . Laminin β2 , a protein concentrated in SBL , could inhibit neurite outgrowth and regulate nerve terminal differentiation and maturation ( Noakes et al . , 1995; Fox et al . , 2007 ) . It is thought to bind directly to and cluster calcium channels , which in turn recruit other presynaptic components ( Nishimune et al . , 2004 ) . In Integrinβ1 muscle-specific mutant mice , nerve terminal arborization and differentiation are abnormal ( Schwander et al . , 2004 ) . Lrp4 and MuSK were shown to be critical for presynaptic differentiation independently ( Kim and Burden , 2008; Yumoto et al . , 2012; Wu et al . , 2012b ) . Previously , we demonstrated that Ctnnb1 in muscle is critical for the differentiation of motor nerve terminals ( Li et al . , 2008 ) . In this paper , we dissected out which functions of muscle Ctnnb1 is critical by an in vivo transgenic approach . We showed that muscle overexpression of the Ctnnb1 mutant without the TAD was unable to rescue the presynaptic deficits of Ctnnb1 mutation , indicating that the transcription regulation is critical for muscle Ctnnb1 to control presynaptic differentiation . On the other hand , the cell-adhesion function of Ctnnb1 is dispensable . These results suggest that muscle Ctnnb1 is likely to regulate presynaptic differentiation by controlling the expression of muscle-derived proteins . This notion was supported by the observations that Ctnnb1 and Wnt signaling reporter were higher in synaptic nuclei at the NMJ than those in extra-synaptic areas . We reasoned that such a protein is secretable or has a transmembrane domain and its expression is down-regulated in Ctnnb1 mutant muscle . A screen for proteins that fulfill these criteria led to the identification of Slit2 . We showed , first , that Ctnnb1 associates with Tcf/Lef cis-elements in the promoter region of the Slit2 gene . Second , Slit2 expression in muscle cells is promoted by elevating Ctnnb1 . Slit2 was concentrated at the NMJ . Third , transgenic mice expressing Slit2 specifically in the muscle were able to diminish presynaptic deficits by Ctnnb1 mutation , including poor innervation , reduced vesicle density and active zone , and depressed mEPP . Finally , Slit2 immobilized on beads was able to induce the synaptophysin puncta of axons of spinal cord explants . Together , these observations suggest that Slit2 serves as a factor utilized by muscle Ctnnb1 to direct presynaptic differentiation . Slit2 is a member of the Slit family of secreted glycoproteins , which serve as repulsive cues for axons of various neurons ( Nguyen-Ba-Charvet et al . , 2002; Shu et al . , 2003; Marillat et al . , 2004; Sabatier et al . , 2004; Hussain et al . , 2006 ) , as regulators to promote axonal and dendritic branching ( Brose et al . , 1999; Wang et al . , 1999; Nguyen Ba-Charvet et al . , 2001; Whitford et al . , 2002; Miyashita et al . , 2004 ) , and as repellants for neuron migration ( Wu et al . , 1999 ) . A previous work showed that motor neurons use Slit2 to promote their axon fasciculation by an autocrine or juxtaparacrine mechanism ( Jaworski and Tessier-Lavigne , 2012 ) . In Slit2 mutant mice , motor nerves prematurely defasciculate into smaller bundlers in various muscles , unlike those in wild type that stay in compact fascicle until they reach the middle of muscle fibers . NMJ morphology at gross anatomic level appears to be normal in Slit2 mutant mice ( Jaworski and Tessier-Lavigne , 2012 ) . We show that Slit2 is produced by muscle cells in a manner dependent on Ctnnb1 . Remarkably , Slit2 , when immobilized on beads , was synaptogenic—to promote the formation and enlargement of synaptophysin-positive punta . Soluble Slit2 did not have this effect , suggesting that the effect requires local , high concentration as many synaptogenic proteins do ( Dean et al . , 2003; Yumoto et al . , 2012 ) . This high concentration in vivo may be achieved by its enrichment at the NMJ , probably due to the involvement of the laminin-G domain of Slit2's extracellular region . The laminin-G domain is known to mediate the SBL immobilization of many proteins including agrin , α-dystroglycan , perlecan , and neurexin ( Timpl et al . , 2000; Chedotal , 2007 ) . The molecular mechanism of Slit2's synaptogenic effect warrants future investigation . Interestingly , the extracellular region of Slit2 has four leucine-rich repeats ( LRR ) -11-residue sequence rich in leucines at defined positions ( LxxLxLxxNxL , x being any amino acid ) . The LRRs have been conserved in some synaptogenic proteins ( de Wit et al . , 2011 ) . The presynaptic deficits in ACTA1-Ctnnb1−/− mice can be grouped into two . One includes mislocation of the primary branches of the phrenic nerve and secondary branch arborization and length , while the other consists of synaptic vesicle number reduction , fewer active zones , and reduced mEPP frequency that was due to reduced release probability ( Li et al . , 2008 ) . This observation suggests that the ‘trophic’ activity of limb buds or muscles identified in the classic limb bud removal experiment is mediated by a Ctnnb1-independent mechanism . Interestingly , Slit2 muscle expression was able to rescue the second group of presynaptic deficits . Besides Slit2 , several other secretable factors were found lower in ACTA1-Ctnnb1−/− muscles . Their function in NMJ formation may be limited or opposite of the hypothetic factor downstream of muscle Ctnnb1 . For example , Mmp9 and Cxcl15 mutant mice are viable and presumably have normal NMJ function ( D'Arcangelo et al . , 1996; Coussens et al . , 2000; Chen et al . , 2001 ) , excluding their involvement in the Ctnnb1 regulation . Hgf was shown to be necessary for motor neuron survival ( Wong et al . , 1997; Yamamoto et al . , 1997 ) ; however , the number of motor neurons was not reduced in ACTA1-Ctnnb1−/− mice or in mice overexpressing Ctnnb1 in muscles ( Wu et al . , 2012a ) . β1-Integrin in muscle is known to be required for NMJ formation ( Schwander et al . , 2004 ) . In particular , axons in β1-Integrin mutant mice failed to respect muscle boundaries and grew onto the tendon organ . However , they do not have nerve defasciculation defects as observed in Ctnnb1-mutant mice . These results do not support a possible involvement of these factors in muscle Ctnnb1 regulation of presynaptic differentiation . It is worthy pointing out that Slit2 overexpression in muscles has no effect on deficits involving primary and secondary branches . Therefore , presynaptic differentiation may be regulated by multiple factors derived from the skeletal muscle . Recent evidence suggests that Wnt signaling regulates NMJ formation . Some Wnts can stimulate while others inhibit agrin-induced AChR clustering ( Henriquez et al . , 2008; Strochlic et al . , 2012; Zhang et al . , 2012; Barik et al . , 2014b ) . The receptor of Wnts at the NMJ may include MuSK that has a CRD domain homologous to that in the Wnt receptor Frizzled ( Masiakowski and Yancopoulos , 1998 ) and LRP4 , a member of the LDLR family whose members , like LRP5/6 , serve are Wnt receptors ( Tamai et al . , 2000 ) . In zebrafish , MuSK is thought to interact with Wnt11r to regulate NMJ formation ( Zhang et al . , 2004; Jing et al . , 2009 ) . Moreover , many Wnt signaling molecules have been implicated in NMJ development , including Dvl and APC ( Luo et al . , 2002; Wang et al , 2003 ) . The deletion of the CRD domain of MuSK in mice causes both pre- and post-synaptic defects of the neuromuscular junction ( Messéant et al . , 2015 ) . Whether Ctnnb1 regulation of presynaptic differentiation is downstream of Wnts is unclear . There are 19 Wnt ligands in mice , which makes it difficult to investigate their function by mutant mice . Perhaps due to functional redundancy , mice lacking Wnt4 or Wnt11 , an isoform most homologous to Wnt11r in zebrafish , were able to form NMJ ( Banerjee et al . , 2011; Strochlic et al . , 2012 ) .
Ctnnb1 conditional KO mice were generated by crossing floxed Ctnnb1 ( Ctnnb1fl/fl ) ( Jackson Laboratory; stock #:004152 ) with ACTA1-Cre transgenic mice that were generously provided by Dr . Melki , to generate the ACTA1-Ctnnb1−/− mice which lacked Ctnnb1 in skeletal muscles . For the rescue experiments , three loxP-STOP-loxP ( LSL ) -Ctnnb1 transgenic mice ( wild-type Ctnnb1; Ctnnb1TV-AA and Ctnnb1∆TAD ) were generated . Ctnnb1TV-AA and Ctnnb1∆TAD ( lacking amino acids 684–781 ) were generated using PCR . The HA tag at the C-terminus was added by subcloning the transgenes into the SalI and XbaI sites of pKH3 . HA-tagged wild type and mutants were subcloned into the XhoI and NotI sites of pCCALL2 ( loxP-STOP-loxP ) vector to generate LSL-Ctnnb1 or mutant mice . LSL-transgenic mice were crossed with Ctnnb1fl/+ to generate Ctnnb1fl/+;LSL-Ctnnb1 or mutant mice . To generate ACTA1-Slit2 mice , Flag-Slit2 was subcloned into pBSX-ACTA1 as described previously ( Monks et al . , 2007 ) . Axin2-nlacZ mice were obtained from Jackson laboratory ( stock #:009120 ) . DNA sequences of all transgenes were validated by sequencing . Linearized DNA was microinjected into FVB blastocysts , which were introduced into pseudo-pregnant recipient females . Mice were genotyped by PCR analysis of tail biopsy DNA . Tail DNA was digested with buffer A ( 25 mM NaOH , 0 . 2 mM EDTA ( ethylenediaminetetraacetic acid ) ) for 45 min at 100°C and neutralized with buffer B ( 40 mM Tris–HCl , pH 8 . 0 ) for genotyping . Mice were housed in cages that were maintained in a room with 12-hr light/dark cycle with ad libitum access to water and rodent chow diet ( Diet 1/4″ 7097 , Harlan Teklad ) . P0 pups of either sex were analyzed , unless otherwise indicated . ‘Material and method’ have been approved by the Institutional Animal Care and Use Committee ( IACUC ) of Georgia Regents University . Muscles were lysed in modified RIPA buffer ( 50 mM Tris–HCl , pH 7 . 4 , 150 mM NaCl , 1% NP-40 , 2% sodium dodecyl sulfate ( SDS ) , 2% deoxycholate , 1 mM PMSF ( phenylmethanesulfonylfluoride ) , 1 mM EDTA , 5 mM sodium fluoride , 2 mM sodium orthovanadate , and protease inhibitors including 1 mM phenylmethylsulfonyl fluoride , 1 μg/μl pepstatin , 1 μg/μl leupeptin , and 2 μg/ml aprotinin ) . After centrifuging at 10 , 000 RPM at 4°C , supernatant was designated as lysates . Protein concentration was measured using Pierce BCA kit . Samples ( 50 μg of protein ) were resolved by SDS-PAGE and transferred to nitrocellulose membrane , which was incubated in 5% milk in phosphate buffer saline ( PBS ) -0 . 3%Tween20 overnight at 4°C and then with primary antibodies in 2% milk in PBS-Tween buffer: Ctnnb1 ( 1:2000 , Cell Signaling #9562 ) ( Danvers , MA ) ; Slit2 ( 1:1500 , Abcam , ab7665 ) ( San Francisco , CA ) ; HA ( 1:2000 , Sigma , H9658 ) ( St . Louis , MO ) ; Flag ( 1:1500 , Sigma , F3165 ) ; GFP ( 1:1000 , Sigma , G1546 ) ; Cre ( 1:400 , Abcam , Ab24607 ) ; β-actin ( 1:4000 , Sigma , A5441 ) . After washing , the membrane was incubated with PBS-Tween buffer containing HRP-conjugated goat anti-mouse and rabbit IgG from Pierce ( 1:5 , 000 , PI-31430 [anti-mouse] , PI-31460 [anti-rabbit] ) . Immunoreactive bands were visualized by using enhanced chemiluminescence ( Life Technologies , Grand Island , NY ) . Quantitative densitometry of captured images was analyzed with Image J ( NIH ) , as described before ( Barik et al . , 2014a ) . Diaphragms were stained whole mount as previously described with modification ( Li et al . , 2008; Wu et al . , 2012a , 2012b ) . Briefly , entire diaphragms with ribs were freshly dissected from mice and fixed in 4% paraformaldehyde ( PFA ) in PBS ( pH7 . 3 ) at 4°C for 3 days , rinsed with PBS ( pH 7 . 3 ) at 25°C , and incubated with 0 . 1 M glycine in PBS for 1 hr at room temperature . The pH of PBS including PFA was critical for proper staining . After rinse with 0 . 5% Triton X-100 in PBS , diaphragms were incubated in the blocking buffer ( 5% bovine serum albumin [BSA] and 1% Triton X-100 in PBS ) for 3 hr at room temperature . Tissues were then incubated with primary antibodies in the blocking buffer overnight at room temperature on a rotating shaker . After washing 3 times for 1 hr each with 0 . 5% Triton X-100 in PBS , tissues were incubated with Alexa Fluor 488 or Alexa Fluor 633-conjugated antibody to rabbit or mouse IgG ( 1:500 , Invitrogen , Carlsbad , CA ) and rhodamine-conjugated α-BTX/alexa-488 conjugated α-BTX ( 1:2000 , Invitrogen , Carlsbad , CA ) overnight at room temperature . After washing 3 times for 1 hr each with 0 . 5% Triton X-100 in PBS and rinsing once with PBS , tissues were flat-mounted in Vectashield mounting medium ( H-1000 , Vector laboratories , Burlingame , CA ) . Z-serial images were collected with a Zeiss confocal laser scanning microscope ( LSM 510 META 3 . 2 ) and collapsed into a single image . For quantitative analysis , images were captured at a subsaturating amplifier gain without modification and quantified using LSM Image Browser and FIJI ( NIH ) software ( Wu et al . , 2012a , 2012b; Barik et al . , 2014a ) . Electron microscopy was performed as described previously ( Wu et al . , 2012a , 2012b; Barik et al . , 2014a ) . Entire diaphragms were lightly stained with R-BTX ( 1:1000 , in ice cold PBS at 4°C ) to mark the central region where NMJs are enriched . The regions were dissected with a micro scalpel ( Harvard apparatus , #PY2 56-5673 ) under a Leica fluorescent dissection scope and fixed ( blocks of approximately 4 mm × 4 mm ) in 2% glutaraldehyde and 2% PFA in 0 . 1 M PBS for overnight at 4°C . Tissues were further fixed in sodium cacodylate-buffered ( pH 7 . 3 ) 1% osmium tetroxide for 1 hr at 25°C . Fixed tissues were washed 3 times with PBS and subjected to dehydration through a series of ethanol: 30% , 50% , 70% , 80% , 90% , and 100% . After 3 rinses with 100% propylene oxide , samples were embedded in plastic resin ( EM-bed 812 , EM Sciences ) . Serial sections ( 1–2 μm ) were stained with 1% Toluidine Blue to identify motor nerves and were cut into ultra-thin sections . Alternate longitudinal sections were not chosen to avoid duplicity of obtaining images from same terminals . They were mounted on 200-mesh unsupported copper grids and stained with a solution containing 3% uranyl acetate , 50% methanol , 2 . 6% lead nitrate , and 3 . 5% sodium citrate ( pH 12 . 0 ) . Electron micrographs were taken using a JEOL 100CXII operated at 80 KeV . Recording was performed as described previously ( Li et al . , 2008; Wu et al . , 2012a , 2012b; Barik et al . , 2014a ) . Neonatal mice diaphragms with ribs and intact phrenic nerves were dissected in oxygenated ( 95% O2/5% CO2 ) Ringer's solution ( 136 . 8 mM NaCl , 5 mM KCl , 12 mM NaHCO3 , 1 mM NaH2PO4 , 1 mM MgCl2 , 2 mM CaCl2 , and 11 mM D-glucose , pH 7 . 3 ) and pinned on Sylgard gel in a dish perfused with oxygenated Ringer's solution . To measure mEPP , microelectrodes , 20–50 MΩ when filled with 3 M KCl , were pierced into the center of muscle fibers with the resting potential between −45 and −55 mV . To evoke end-plate potentials , phrenic nerves were stimulated by a suction electrode with suprathreshold square pulses ( 0 . 1 ms ) using Master-8 ( A . M . P . I , Jerusalem , Israel ) . Data were collected with axonpatch 200B amplifier ( Molecular Devices , Sunnyvale , CA ) , digitized with Digidata 1322A ( Molecular Devices , Sunnyvale , CA ) , and analyzed using pClamp 9 . 2 ( Molecular Devices , Sunnyvale , CA ) . Mouse muscle C2C12 myoblasts were propagated and induced to form myotubes as described previously ( Luo et al . , 2003 ) . C2C12 myoblasts were transfected with lipofectamine 2000 ( Invitrogen , 11 , 668-019 ) with a modified protocol . C2C12 myoblasts , at 70–80% confluence , were rinsed once with serum-free medium before transfection because serum appeared to reduce transfection efficiency . After complete aspiration of the medium , myoblasts were incubated with a mixture of DNA , lipofectamine , and serum-free medium for 8 hr when the medium was changed to the growth medium . The DNA:lipofectamine ratio in the mixture was 1 μg:2 μl . The optimal volume of the mixture for 24-well dishes was 200 μl medium per well that contained 2 μg plasmid DNA . HEK293 cells were cultured and transfected as previously described ( Luo et al . , 2008; Wu et al . , 2012b ) . ChIP assays were performed according to the protocol provided by Upstate Biotechnology ( Kim et al . , 2005 ) . C2C12 myotubes were cultured on gelatin-coated 6-well dish , transfected with HA-Ctnnb1 , were washed twice with PBS and then incubated in 1% formaldehyde in PBS at room temperature for 10 min . After washing twice with PBS , cells were scraped in PBS containing protease inhibitors , centrifuged at 2000 rpm at 4°C , and resuspended in SDS lysis buffer containing 1% SDS , 10 mM EDTA , 50 mM Tris–HCl ( pH 8 . 0 ) plus protease inhibitors . Cells were sonicated by Sonic Dismembrator model 100 ( Fisher , Suwanee , GA ) , strength 3 , for 15 s 3 times , to shear chromosome DNA . After centrifugation at 13 , 000 at 4°C , the sonicated mixture was diluted 10-fold with ChIP dilution buffer containing 0 . 01% SDS , 1 . 1% Triton X-100 , 1 . 2 mM EDTA , 16 . 7 mM Tris–HCl ( pH 8 . 1 ) , and 167 mM NaCl . The mixture was precleaned once with salmon sperm DNA-protein A-agarose beads at 4°C for 1 hr . The resulting supernatant was incubated with anti-Ctnnb1 antibody at 4°C overnight , when salmon sperm DNA-protein A-agarose beads ( 50 μg/μl ) was added . The beads/mixture was incubated for another hour at 4°C and washed sequentially with a low-salt wash buffer containing 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , 20 mM Tris–HCl ( pH 8 . 1 ) , and 150 mM NaCl; a high-salt wash buffer containing 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , 20 mM Tris–HCl ( pH 8 . 1 ) , and 500 mM NaCl; an LiCl wash buffer containing 0 . 25 M LiCl , 1% NP-40 , 1% deoxycholic acid , 1 mM EDTA , and 10 mM Tris–HCl ( pH 8 . 1 ) ; and a wash with TE buffer . Beads were then incubated twice with 250 μl of the elution buffer ( 1% SDS , 0 . 1 M NaHCO3 ) for 15 min at room temperature to elute bound DNA . Combined eluates were added to 20 μl of 5 M NaCl to reverse cross-links by heating at 65°C for 4 hr . After incubation with 10 μl of 0 . 5 M EDTA , 20 μl of 1 M Tris–HCl ( pH 6 . 5 ) , and 2 μl of 10 mg/ml proteinase K ( 1 hr at 45°C ) , DNA was recovered by phenol-chloroform extraction and ethanol precipitation and used as template for PCR . Primers for the two TCF4/ LEF1-binding regions were as follows; Slit2 ( primer1 ) - 5′-GTCCC CTTTA GGATC GCG-3′/5′-CAGCG GGAGA ACGAG G-3′ and Slit2 ( primer2 ) -5′-AGAGA AGGTT GAAAA CACTA CTCCC-3′/5′-TGAAA TAGAT CTGCC TCCGT G-3′ . Total RNA was isolated from muscles using Trizol ( Invitrogen ) and purified using the RNeasy mini kit ( Qiagen ) . Quantitative RT-PCR was performed as described previously ( Wu et al . , 2012a ) . Equal amounts of total RNA ( 1 μg ) were reverse transcribed by random hexamer primers using the Maxima Enzyme Mix ( Fermentas ) . Quantitative PCRs were run in a PTC-200 Peltier Thermal Cycler ( Bio-Rad MJ Research ) using SYBR Green/ROX ( Fermentas ) . The primers for specific genes are listed in Supplementary file 1 . Spinal cord explant culture from E12 . 5 embryos was performed as previously described ( Wang and Marquardt , 2012 ) . In short , pregnant mouse was euthanized at E12 . 5 after anesthesia by isoflurane and subsequent cervical dislocation . Embryos along with amniotic sacs were dissected and placed in ice-cold PBS . Spinal cords were dissected out of the embryos exposing ventral motoneuron columns . Ventral horns were dissected out by a scalpel and cultured on laminin ( Invitrogen #23017-15 ) coated coverslips in neurobasal medium ( catalog #21103-049; Invitrogen ) supplemented with B27 ( catalog #17504-044; Invitrogen ) , 600 μM/ml-glutamine ( catalog #25- 005-CI; Cellgro ) , BDNF ( 2 ng ml−1 ) , GDNF ( 2 ng ml−1 ) , CTNF ( 2 ng ml−1 ) , NGF ( 1 ng ml−1 ) ( Sigma ) , and penicillin-streptomycin ( catalog #30-003-CI; Cellgro ) . Every alternate day , half of the medium was replaced by freshly prepared medium . 6 days after plating , the explants are subjected to incubating with Slit2-conjugated beads ( see below ) . HEK293 cells were transfected with Flag-Slit2 or control empty Flag vector , to produce Flag-Slit2 and control conditioned media , respectively . Biotin-conjugated mouse monoclonal antibody ( 2 μl ) ( anti-Flag BioM2 , Sigma , F9291 ) was conjugated with 50 μl of FluoSpheres NeutrAvidin-labeled Microspheres ( 1 . 0 µm , red fluorescent [580/605] , F-8775 , Invitrogen ) by incubation in PBS for 4 hr at 4°C and then washed with ice-cold PBS , 3 times . Beads were re-suspended in 200 μl of ice-cold PBS with protease inhibitors and incubated with Flag-Slit2 or control conditioned media ( 200 μl ) overnight on a rotating shaker at 4°C . Flag-Slit2-conjugated neutravidin microspheres were isolated by spinning at 5000 rpm at 4°C . Microspheres were washed 3 times with ice-cold PBS and diluted in explant culture media ( 1:100 ) and incubated with spinal cord explants for 24 hr before analysis . Dorsal horn explants were fixed 4% ice-cold PFA for 20 min at room temperature and washed twice ( 5 min each ) with 1 ml PBS . Explants were then permeabilized by 1 ml blocking solution ( PBS with 0 . 3% Triton X-100 , 5% BSA , and 3% goat serum ) for 5 min at room temperature and incubated with primary antibodies in blocking solution for 2 days at 4°C . Explants were rinsed 3 times with 1 ml PBS , 5 min each , and incubated with secondary antibodies in blocking solution for 90 min at room temperature . After washing thrice with 1 × PBS , 5 min each , coverslips were mounted on glass slides and covered with Vectashield mounting medium ( H-1000; Vector Laboratories ) . Images were obtained by confocal scanning microscopy . FIJI ( NIH ) was used to measure and quantify synaptophysin puncta number and size . Two-tailed student t-test was used to compare data between two groups . One-way ANOVA ( analysis of variance ) was used to compare data between multiple groups , and Bonferroni's Multiple Comparison Test was used to determine the significance . Unless otherwise indicated , data were expressed as mean ± SEM , and considered statistically significant if p value was less than 0 . 05 .
Neuromuscular junction formation requires proper interaction between motoneurons and muscle cells . β-Catenin ( Ctnnb1 ) in muscle is critical for motoneuron differentiation; however , little is known about the relevant retrograde signal . In this paper , we dissected which functions of muscle Ctnnb1 are critical by an in vivo transgenic approach . We show that Ctnnb1 mutant without the transactivation domain was unable to rescue presynaptic deficits of Ctnnb1 mutation , indicating the involvement of transcription regulation . On the other hand , the cell-adhesion function of Ctnnb1 is dispensable . We screened for proteins that may serve as a Ctnnb1-directed retrograde factor and identified Slit2 . Transgenic expression of Slit2 specifically in the muscle was able to diminish presynaptic deficits by Ctnnb1 mutation in mice . Slit2 immobilized on beads was able to induce synaptophysin puncta in axons of spinal cord explants . Together , these observations suggest that Slit2 serves as a factor utilized by muscle Ctnnb1 to direct presynaptic differentiation .
Motor nerves are like electrical wires that connect our spinal cord to the muscles in our body . These nerves communicate with muscles across a connection called the neuromuscular junction . To first form a neuromuscular junction , the motor nerves and muscles each produce molecular cues that tell each other to do their part to build a connection . Beta-catenin in the muscle is known to regulate motor nerve development . However , beta-catenin has two different roles: it helps to coordinate whether neighboring cells stick together , and it can regulate which genes are ‘transcribed’ to produce proteins . It was not known which of these roles is necessary for forming neuromuscular junctions . Wu , Barik et al . now investigate this question by creating mice with mutant forms of beta-catenin in their muscles . Some mice had muscle beta-catenin that could not help cells stick together , and others had beta-catenin that could not control gene transcription . Only mutations that affected the ability of beta-catenin to control transcription caused abnormalities in the neuromuscular junction . However , these problems could be fixed by adding either normal beta-catenin or the mutant form that cannot help cells stick together . Wu , Barik et al . then used molecular tools to explore which genes are turned on by beta-catenin . The experiments showed that beta-catenin causes muscle fibers to produce a protein called Slit2—a developmental cue that controls where neurons grow . Furthermore , the neuromuscular junction defects found in mice without beta-catenin in their muscles could be reduced by making the muscle fibers produce more Slit2 . However , not all defects in beta-catenin mutant mice are rescued by Slit2 . Future research is needed to identify other beta-catenin-controlled signals and to determine whether such a pathway is altered in neuromuscular disorders .