Article |
Address correspondence to Christine E. Beattie, Center for Molecular Neurobiology, The Ohio State University, 115 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. Tel.: (614) 292-5113. Fax: (614) 292-5379. email: beattie.24{at}osu.edu
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Abstract |
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Key Words: SMA; SMN; motoneurons; zebrafish; morpholino
Abbreviations used in this paper: AChR, acetylcholine receptor; CaP, caudal primary; MO, morpholino oligonucleotide; SMA, spinal muscular atrophy; SMN, survival motor neuron; VeLD, ventral longitudinal descending.
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Introduction |
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SMA, an autosomal recessive disorder, is the leading hereditary cause of infant mortality (Roberts et al., 1970) and is characterized by loss of motoneurons in the spinal cord (Crawford and Pardo, 1996; Melki, 1997). The disease results from low levels of the protein encoded by the survival motor neuron (SMN) gene. Although SMN is expressed in all cell types, motoneurons are specifically affected in SMA, indicating their sensitivity to low SMN levels (Coovert et al., 1997; Lefebvre et al., 1997; Monani et al., 2000). Humans have two copies of the SMN gene, SMN1 and SMN2, which differ in a single base change in a splice enhancer site for exon 7 of SMN2 (Lorson et al., 1999; Monani et al., 1999; Cartegni and Krainer, 2002). Thus, SMN1 produces a majority of full-length transcript, whereas SMN2 generates mostly transcripts lacking exon 7, although some full-length transcript is produced (Lefebvre et al., 1995). SMN protein lacking exon 7 does not oligomerize effectively (Lorson and Androphy, 1998) and appears to be unstable and rapidly degraded (Lorson and Androphy, 2000). Thus, mutations in SMN1, but retention of the SMN2 gene, results in reduced protein levels and ultimately SMA (Lefebvre et al., 1995, 1997; Coovert et al., 1997).
The 38-kD SMN protein is ubiquitously expressed and localizes to both the cytoplasm and nucleus (Liu and Dreyfuss, 1996; Coovert et al., 1997; Lefebvre et al., 1997). In the nucleus, SMN localizes to structures termed gems, which overlap or are in close proximity to coiled bodies (Liu and Dreyfuss, 1996; Young et al., 2000a). It has been termed the master RNA assembler and, in particular, has been shown to be important in assembly of snRNP particles (for review see Terns and Terns, 2001). SMN also binds to the hn-RNP-R, which is involved in RNA editing and mRNA transport (Rossoll et al., 2002). Recent data shows that hn-RNP-R colocalizes with SMN in distal axons of embryonic motoneurons (Jablonka et al., 2001; Rossoll et al., 2002). SMN also has been shown to localize in the growth cones and branch points of developing neurons (Jablonka et al., 2001; Fan and Simard, 2002; Zhang et al., 2003). Ultimately, however, the function of SMN in relation to SMA pathology and etiology remains unclear.
To further analyze SMN function, animal models of SMA have been generated (Schrank et al., 1997; Hsieh-Li et al., 2000; Monani et al., 2000; Cifuentes-Diaz et al., 2002; Monani et al., 2003). In contrast to humans, mice have only one Smn gene, which is equivalent to human SMN1 (DiDonato et al., 1997; Viollet et al., 1997). Complete loss of this gene results in an embryonic lethal phenotype (Schrank et al., 1997). Introduction of one or two copies of human SMN2 rescues the embryonic lethal phenotype and results in mice with severe SMA (Hsieh-Li et al., 2000; Monani et al., 2000), whereas 816 copies of SMN2 completely rescue the SMA phenotype (Monani et al., 2000). Although both severe and mild SMA mice ultimately exhibit motoneuron cell body reduction (Monani et al., 2000, 2003), no early morphological or biochemical abnormality of the motoneurons has been reported.
A model of SMA in zebrafish has the potential to elucidate the effect of decreased Smn levels on motoneuron development in vivo. At 24 h, there are three well-characterized primary motoneurons per spinal cord hemisegment that innervate either the dorsal, rostral, or ventral region of each myotome (Eisen et al., 1986; for review see Beattie, 2000; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200303168/DC1). Over the next few days, each of the primary motor axons are joined by 2030 secondary motor axons, which form three distinct nerves that innervate the three myotome regions (Myers et al., 1986; Pike et al., 1992). As these axons extend into defined myotome regions, they can be followed in living embryos; thus, perturbations in the organization of these neurons or their axons can be readily detected, followed during development, and quantitated (for review see Beattie, 2000).
We have used antisense morpholino technology to model the effects of low levels of smn in zebrafish. Reducing Smn protein levels in the developing embryo results in motor axonspecific truncations and branches, independent of motoneuron cell death. Moreover, by decreasing Smn levels in single motoneurons, we show that these defects are due to a cell-autonomous function of Smn in motoneurons. These are the first reported morphological abnormalities of motoneuron development in response to low levels of Smn. These data reveal that one of the earliest consequences of Smn protein reduction is severely compromised motor axon outgrowth, indicative of an essential role for Smn in motoneuron development.
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Results |
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Knockdown of Smn causes spinal motor axon defects
Morpholino antisense oligonucleotide "knockdown" technology was performed in zebrafish to decrease the levels of the Smn protein and mimic SMA. Morpholino oligonucleotides (MOs) inhibit the translation of their target mRNA, thereby causing reduced levels of target protein (Nasevicius and Ekker, 2000). Control or smn MO was injected into embryos at the one- to four-cell stage, and then embryos were allowed to develop until the desired time. Western blot analysis showed that smn MOinjected embryos exhibited a 61% decrease in Smn protein at 36 h (Fig. 1, A and B). Approximately 78% (n = 580) of embryos injected with 6 ng of smn MO survived compared with 91% (n = 139) survival when injected with the same concentration of control MO. Embryos died between late gastrulation and early somitogenesis, suggesting essential Smn function during zebrafish development. smn MOinjected embryos that survived did not exhibit gross abnormalities.
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Further knockdown of Smn protein results in increased motor axon branching
It has been shown in human patients and mouse models that the severity of SMA is dependent on the amount of SMN protein (Monani et al., 2000). To investigate if motor axon defects would become more severe when Smn protein was further reduced, 9 ng of MO was injected into embryos. Western blot analysis showed a 77% knockdown of protein (Fig. 2, A and B). In contrast to 89% survival when control MO was injected (n = 164), at this higher dose of smn MO, only 45% (n = 433) of the embryos survived, further suggesting the essentiality of Smn in zebrafish development.
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Motor axon truncations were less evident at higher smn MO doses; in fact at 27 h, there is no significant difference when compared with control MO injections, where 5.6% of sides had truncated motor axons (Table I, Fig. 2 D). Also, all of the smn MO defective sides only had one truncated motor axon (Table II). At 36 h, a similar trend was apparent. 46.9% of 9 ng smn MOinjected embryo sides had at least one truncated motor nerve, which is slightly reduced compared with lower doses of MO (Table I; Fig. 2 G). The average number of truncated motor nerves per side is 2.47, slightly less than at lower doses of MO (Table II). These data suggest that further reduction of Smn protein levels results in more severe motor nerve branching defects and less severe truncation defects. To confirm that reduction in Smn results in motor axon/nerve defects, we used an additional, nonoverlapping smn MO and observed similar defects (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200303168/DC1), although this 5' UTR MO was less efficient than the original MO described (Table SI, available at http://www.jcb.org/cgi/content/full/jcb.200303168/DC1).
Aberrant motor nerves remain defective over time
To determine the dynamics of the axon defects observed in smn MOinjected embryos, we analyzed motor nerves over several days using gata2GFP transgenic zebrafish (Meng et al., 1997). In this transgenic line, secondary motoneuron cell bodies and ventrally projecting axons express GFP starting at 33 h. 9 ng of smn MO or control MO was injected into gata2GFP embryos, and ventral motor nerves were visualized from 36 to 74 h. smn MO was still effective in knocking down Smn protein at later time points, as evidenced by Western blot analysis performed at 74 h, which shows a 88% reduction in Smn protein (Fig. 3, A and B).
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Knockdown of smn also causes aberrant dorsal projecting motor nerves
To determine whether other spinal motor axons were affected by decreased levels of Smn protein, we analyzed dorsal motor nerves in islet1GFP transgenic zebrafish. Among other neuronal types, this transgenic line has GFP-expressing secondary motoneuron cell bodies and dorsally projecting axons (Fig. 4 A; Higashijima et al., 2000). 9 ng of smn MO or control MO was injected into islet1GFP transgenic embryos, and dorsal nerves were analyzed at 74 h. Like ventral motor nerves, dorsally projecting motor nerves exhibited truncation and branching defects as a result of smn knockdown (Fig. 4, B and C). Branching is also more prominent at higher doses of smn MO in these nerves. 69.1% (n = 178) of sides of smn MOinjected embryos had at least one branched dorsal projecting motor nerve, whereas control MOinjected embryos had only 8.0% of sides with a branched dorsal nerve (n = 50). In contrast, 32.6% (n = 178) of sides of smn MOinjected embryos had at least one truncated nerve, whereas control MOinjected embryos had no truncated dorsal projecting nerves (n = 50). These defects are consistent with those seen in ventral projecting motor nerves, suggesting that dorsal projecting motoneurons are also affected by reduction of Smn protein.
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CaP motoneurons iontophoresed with 1 µM control MO exhibited no axon defects (n = 10; Fig. 9 A). In contrast, CaP motoneurons, iontophoresed with smn MO when their growth cones were at the first intermediate target, were defective 76.9% of the time (n = 13; Fig. 9, B, C, and D). The defects observed, axon truncations and branches, were consistent with defects seen when the entire embryo was injected with smn MO. CaP motoneurons were also iontophoresed with smn MO when their growth cones were further ventral at the second intermediate target. Only 62.5% of these motor axons (n = 8; Fig. 9 D) were defective, suggesting that the longer the Smn protein is reduced during outgrowth, the more likely pathfinding errors, such as truncations and branches, will occur. The incomplete penetrance of these defects may suggest variability in the phenotype, as is seen when the entire embryo is injected with smn MO.
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Discussion |
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The motor axon defect in smn knockdown embryos
Embryos with decreased Smn levels show defects in motor axon outgrowth and pathfinding during the first three days of development. Defects were rarely seen in the proximal axon extending from the spinal cord to the first intermediate target, suggesting that initiation of axon outgrowth and pathfinding along the common pathway are unaffected by low Smn levels. Secondary motoneurons also displayed branching and truncation defects that continued up to 3 d. As primary and secondary motor axons fasciculate to form nerves, we cannot be certain that the defects observed at 3672 h are defasciculation of the nerve or true branching of the axons. As branching in single CaP axons is seen, this suggests that true branching does occur. Although the motor axon projections are aberrant, these axons still colocalized with AChR, indicating that functional synapses had formed.
The motor axon branching and truncation defects were initially discovered upon injection of 6 ng of smn MO into zebrafish embryos. When a higher dose of MO, 9 ng, was used, there was a significant increase in the amount of motor axon branching and an apparent decrease in truncations. Analysis of embryos with GFP-expressing motor nerves, however, revealed that truncations were still occurring but were followed by branching, thus obscuring the truncation phenotype. These data indicate that the less Smn protein translated within the neuron, the greater the likelihood that the axon will branch. Ventral nerve truncations were consistently occurring at the second intermediate target. It is believed that intermediate targets are important for growth cones to proceed to their final targets (Tessier-Lavigne and Goodman, 1996). It is possible that in the presence of low Smn, growth cones stall at these locations, suggesting that they still try to assimilate cues. At very low levels of Smn, the growth cone may not even be responding to the intermediate target and branches indiscriminately.
Cell autonomy of Smn
Due to the distribution of SMN protein, it has been difficult to conclusively determine whether the primary defect resides in motoneurons or muscle. In smn MOinjected zebrafish embryos, the motoneurons are defective, but muscle specification, early patterning, and muscle development are normal, indicating that decreasing Smn is critical in motoneurons. We specifically reduced Smn in single motoneurons without affecting Smn levels in other neurons or muscle. Exclusively decreasing Smn in motoneurons recapitulated the motor axon defect seen when Smn was decreased throughout the embryo, indicating that Smn functions cell autonomously. We cannot, however, rule out the possibility that Smn also functions in muscle to affect motor axons.
Smn and motor axons
Recent mouse and human studies suggest that motor axons may be affected before motoneuron loss. Mice exhibiting neuronal depletion of SMN (SmnF7/SmnD7, NSE-Cre+) exhibited a dramatic decrease in motor axons but only a modest decrease in motoneuron cell bodies at postnatal day 15 (Cifuentes-Diaz et al., 2002). Due to the severe reduction in full-length SMN and the relatively late developmental timing of this reduction, however, it is difficult to determine the disease relevance of these defects. Severe SMA mice (Smn-/- with one to two copies of SMN2; Hsieh-Li et al., 2000; Monani et al., 2000) show moderate motoneuron cell body loss, whereas mild SMA mice (A2G missense) show no motoneuron cell body loss several days after birth; mild SMA mice do, however, exhibit a mild reduction in both motoneuron cell bodies and motor axon roots at 3.5 mo (Monani et al., 2003). Motor unit number estimation (MUNE) studies in humans show that presymptomatic individuals exhibit high motoneuron counts that dramatically decrease with onset of the disease (Bromberg and Swoboda, 2002). These results indicate that motoneuron cell body loss is a late feature of the disease. Our data are consistent with these findings and support the idea that axons are affected before cell death.
SMN has also been shown to localize in developing motoneurons. Rat SMN colocalizes with cytoskeletal elements in the spinal motor axons during nervous system maturation (Pagliardini et al., 2000). In primary motoneuron cultures from E14 mice embryos, SMN is enriched in branch points and growth cones (Jablonka et al., 2001). SMN has also been found in growth cones of P19 neurons (Fan and Simard, 2002). Interestingly, SMN interacting protein 1 (SIP1), a protein important in spliceosomal snRNP biogenesis (Fischer et al., 1997), does not coenrich in axon branch points, indicating the possibility of a unique SMN complex and function at these locations (Jablonka et al., 2001). Taken together with our findings of abnormal motor axon branching when Smn is depleted, the data suggest that Smn plays an important function in motor axon development and maintenance.
Although a mechanism by which SMN functions in motoneuron development remains unclear, recent data do give clues. SMN has been shown to interact with profilin (Giesemann et al., 1999), a protein involved in actin polymerization and growth cone motility (Gutsche-Perelroizen et al., 1999; Wills et al., 1999; Kim et al., 2001), processes important for axon outgrowth. Data have also implicated an interaction between SMN and motoneuron-enriched RNA processing molecules, hn-RNP-Q and hn-RNP-R (Rossoll et al., 2002). These proteins play a role in RNA transport, editing, and translation. RNA transport into the axon followed by localized protein translation has been shown to be important for growth cones to proceed past intermediate targets (Brittis et al., 2002). It is possible that SMN in axons and growth cones may be associated with RNP complex assembly associated with transport and/or translation of mRNA. Recent experiments have shown that SMN localizes with cytoskeletal filaments and is actively transported down axons (Zhang et al., 2003), further indicating that SMN may be involved in transport and the resulting translation of important axon guidance cues. It is possible that if SMN is reduced, motor axons cannot respond to cues at intermediate targets by turning on local translation, resulting in axon defects. Our finding that GFP-expressing motor nerves in smn MOinjected embryos are truncated at the second intermediate target is consistent with this idea. The validity of this model, however, will depend on elucidating a direct relationship between Smn and localized protein translation machinery in motor axon growth cones.
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Materials and methods |
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Nomenclature
Consistent with guidelines for different species, the mouse, human, or rat protein is SMN and the gene is SMN (human) or Smn (mouse). In fish, the protein is Smn, and the gene is smn (http://zfin.org/zf_info/nomen.html).
Physical mapping and southern blot analysis
Zebrafish smn primers (5'-GTGATGATTCTGACATTTGG-3' and 5'-CCATCCTCACCTTTCAAAGC-3') were used to map the gene on the LN54 radiation hybrid panel (Hukriede et al., 1999). smn maps to linkage group 5, 3.25 cM from marker fb39c12. Southern blots were performed as previously described (Monani et al., 2000). The smn probe was generated by PCR with the following primer sequences: 5'-GTGATGATTCTGACATTTGG-3' and 5'-GTCTTCAGAGCATCTTCATCC-3'. The zebrafish islet2 gene was used as a control.
Whole-mount in situ hybridization
Zebrafish smn cDNA clone (GenBank/EMBL/DDBJ accession nos. Y17256, AA494875, AA494767, and AF083557) was used to make sense and antisense digoxigenin-labeled riboprobes of 1016 bp (Bertrandy et al., 1999). The sense (Sp6) and antisense (T7) smn riboprobes were synthesized from plasmid linearized with XhoI and HindIII, respectively. Islet2 and MyoD riboprobes were synthesized as previously described (Appel et al., 1995; Weinberg et al., 1996). Whole-mount in situ hybridization protocol (1277073; Roche) was performed as previously described (Thisse et al., 1993).
Antisense MO and synthetic mRNA injections
An antisense MO was designed against the 5' start sequence of the smn gene (Gene Tools, Inc.); 5'-CGACATCTTCTGCACCATTGGC-3'. An additional nonoverlapping MO was also designed 20 bp upstream of the original MO (designated 5' UTR MO); 5'-TTTAAATATTTCCCAAGTCCAACGT-3'. Two- to four-cell *AB, gata2GFP (Meng et al., 1997), or islet1GFP (Higashijima et al., 2000) embryos were injected with
6 or 9 ng of MO in Danieau's solution with phenol red dye according to protocol (Nasevicius and Ekker, 2000). A standard control (Gene Tools, Inc.) MO (5'-CCTCTTACCTCAGTTACAATTTATA-3') was used at 6 or 9 ng for control injections. Synthetic capped human SMN mRNA was produced using mMESSAGE mMACHINE kit (Ambion) according to the manufacturer's instructions. mRNA was produced from previously described plasmids (Le et al., 2000) linearized with Xho1. Approximately 300500 pg of mRNA was coinjected with 9 ng of smn MO.
Immunohistochemistry
Immunohistochemistry and imaging were performed essentially as previously described (Beattie et al., 2000). The following mAbs were used: znp1 (1:100; Melancon et al., 1997), antiacetylated tubulin (1:250; T-6793; Sigma-Aldrich), antislow twitch myosin F59 (1:10; Crow and Stockdale, 1986; Devoto et al., 1996), antifast twitch myosin F310 (1:10; Crow and Stockdale, 1986; Zeller et al., 2002), 3A10 (1:10; Hatta, 1992), and antineurolin zn-5 (1:75; Fashena and Westerfield, 1999). FITC (IgG2A) and TRITC (IgG1) isotype-specific conjugate secondary Abs (108002 and 107003, respectively; Southern Biotechnology Associates, Inc.) were used for fluorescent detection of znp-1 (IgG2A) and F59/F310 (IgG1) mAbs. Cross-sectional analysis was performed by embedding embryos in 1.5% agar/5% sucrose and sectioning on a crytostat at 16 µm. All immunofluorescent images were analyzed using a confocal microscope and photographed using digital imagery (Nikon Optiphot 2; Bio-Rad Laboratories, Inc. MRC 1024) unless otherwise specified.
Western blot analysis
MO-injected embryos were manually dechorionated and deyolked in a slurry of physiologic Ringers (Westerfield, 1995), Ringers ice chips, and protease inhibitors (Complete Mini; 1836170; Roche). Deyolked embryo samples were prepared and Western blots were performed as previously described (Monani et al., 2003). Smn (1:1,000; MANSMA7 or MANSMA21; Young et al., 2000b), antisynaptic vesicle (SV2) protein (1:200; Buckley and Kelly, 1985), and antiHu 16A11 (1:500; A-21271; Molecular Probes; Marusich et al., 1994) mAb were visualized using the ECL Detection Kit (RPN 2109; Amersham Biosciences). Quantification of bands was performed using a densitometer (Shimadzu, Inc.).
Visualization of GFP transgenic zebrafish
Live gata2GFP MO-injected transgenic zebrafish (Meng et al., 1997) were anesthetized in tricaine (A-5040; Sigma-Aldrich) at 36, 52, and 74 h and mounted on a glass coverslip. Individual motor nerves were visualized and imaged using a Photometrics SPOT camera. Images were compiled and edited using Adobe Photoshop® software. MO-injected islet1GFP embryos (Higashijima et al., 2000) were fixed at 72 h in 4% paraformaldehyde for 2 h at room temperature.
TUNEL assay
TUNEL assay was performed according to the manufacturer's protocol on staged embryos (Cole and Ross, 2001). Digoxigenin-labeled dUTP was used to label fragmenting DNA ends (Terminal Transferase Assay; 220582; Roche), and then embryos were sectioned on a crytostat at 16 µm.
Detection of neuromuscular junctions
MO-injected embryos were staged and fixed in 4% paraformaldehyde for 2 h at room temperature and water soaked for 36 h. After collagenase (C-9891; Sigma-Aldrich) treatment, embryos were then incubated in Alexa®594-conjugated -bungarotoxin (10 µg/ml; B13423; Molecular Probes) for 30 min, essentially as previously described (Ono et al., 2001). Embryos were then Ab stained using znp-1 and a FITC-conjugated secondary Ab.
Single cell knockdown of smn
Glass capillary microelectrodes were backfilled with a solution containing 2.5% rhodamine dextran (3,000 MW; Molecular Probes) and smn or control MO. 19.520.5-h embryos were anesthetized in tricaine and mounted in agar on a microslide as previously described (Eisen et al., 1989; Beattie et al., 2000). Individual CaP motoneurons or VeLD interneurons were impaled electrically by oscillating the electrode tip as previously described (Eisen et al., 1989). Solution was added to the cell by iontophoresis. Images were taken with a Photometrics SPOT camera and compiled and edited in Adobe Photoshop®.
Online supplemental material
The supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200303168/DC1. Fig. S1 diagrams primary and secondary motor axon pathfinding in the developing zebrafish. Fig. S2 shows the expression pattern of smn during the development of zebrafish. Fig. S3 shows that an additional 5' UTR smn MO results in similar motor axon defects. Table SI shows the percentage of sides with motor axon/nerve defects using additional 5' UTR smn MO.
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Acknowledgments |
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U.R. Monani is the recipient of a Development Grant from the Muscular Dystrophy Association of America. This research is supported by National Institutes of Health grant RO1NS41649.
Submitted: 26 March 2003
Accepted: 14 July 2003
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