Embryonic motor axon development in the severe SMA mouse - PubMed (original) (raw)

. 2008 Sep 15;17(18):2900-9.

doi: 10.1093/hmg/ddn189. Epub 2008 Jul 3.

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Embryonic motor axon development in the severe SMA mouse

Vicki L McGovern et al. Hum Mol Genet. 2008.

Abstract

Spinal muscular atrophy (SMA) is caused by reduced levels of survival motor neuron (SMN) protein. Previously, cultured SMA motor neurons showed reduced growth cone size and axonal length. Furthermore, reduction of SMN in zebrafish resulted in truncation followed by branching of motor neuron axons. In this study, motor neurons labeled with green fluorescent protein (GFP) were examined in SMA mice from embryonic day 10.5 to postnatal day 2. SMA motor axons showed no defect in axonal formation or outgrowth at any stage of development. However, a significant increase in synapses lacking motor axon input was detected in embryonic SMA mice. Therefore, one of the earliest detectable morphological defects in the SMA mice is the loss of synapse occupation by motor axons. This indicates that in severe SMA mice there are no defects in motor axon formation however, we find evidence of denervation in embryogenesis.

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Figures

Figure 1.

Figure 1.

Motor axon development in the severe SMA embryo. Overall morphology of motor axons at the brachial plexus of the forelimb (A–D), lumbosacral plexus of the hindlimb (E–H), and intercostal muscles in the thoracic region (I–L) of e12.5 embryos is normal in SMA embryos (C, G, K) as compared with normal (A, E, I) and carrier (B, F, J) littermate controls. The phrenic nerve of the diaphragm is also patterned correctly in e15.5 SMA embryos (O) when compared with normal (M) and carrier (N) littermate controls. In (A)–(L) anterior is to the top and proximal is to the left. The blue square in each of schematic drawing (B, H, L, P) indicates the location imaged in each tissue. Scale bar represents 300 µm for each micrograph.

Figure 2.

Figure 2.

Axonal outgrowth and NMJ bandwidth in SMA mice. (A, B) No difference in the length of motor axon outgrowth was identified in the intercostal region (T4–T11) of e10.5 carrier (A) and SMA (B) littermates. No increase in the width of the end plate band was observed in PND03 diaphragm muscle between SMA (C) and normal (D) mice as assayed by alpha-bungarotoxin staining. Furthermore, the width of the end plate band was similar in carrier and SMA animals in the intercostal muscles at e18.5 (E, F respectively) and at PND02 (G, H respectively). Scale bar: (A–D) 300 µm; (E–H) 100 µm.

Figure 3.

Figure 3.

Axonal swellings present in severe SMA mice. Motor axons of severe SMA mice (B, D, F) reveal swellings rarely seen in normal littermate controls (A, C, E). The phrenic nerve of PND03 diaphragm muscles (B, F) as well as the nerves innervating the intercostal muscles of e18.5 embryos (D) display several beads in the SMA tissue (arrowheads) that are seldom seen in normal controls (A, C, E). (E, F) Neurofilament antibody staining of PND02 diaphragm muscle also reveals the axonal swellings in SMA neurons (F) that are not often found in normal animals (E). Scale bar represents 50 µm for each micrograph.

Figure 4.

Figure 4.

Unoccupied AChR clusters are found in the SMA animal at e18.5. AChR clusters in the intercostal muscles of e18.5 normal (A–C) carrier (D–F) and SMA (G–I) littermates. GFP staining reveals the motor neuron axons innervating the intercostal muscles in (A), (D), (G), alpha-bungarotoxin labeling of AChR clusters in (B), (E), (H), and merged confocal images (C, F, I). All AChR clusters are fully innervated in normal and carrier animals while most AChR clusters are unoccupied in the SMA animal (arrowheads). One cluster is partially innervated as indicated by the arrow. Scale bar represents 50 µm in each micrograph.

Figure 5.

Figure 5.

Unoccupied AChR clusters are found in the SMA animal at PND02. AChR clusters in the intercostal muscles of PND02 normal (A–C) carrier (D–F) and SMA (G–I) littermates. GFP staining reveals the motor neuron axons innervating the intercostal muscles in (A), (D), (G), alpha-bungarotoxin labeling of AChR clusters in (B), (E), (H), and merged confocal images (C, F, I). All AChR clusters are fully innervated in normal and carrier animals while most AChR clusters are unoccupied in the SMA animal (arrowheads). Scale bar represents 50 µm in each micrograph.

Figure 6.

Figure 6.

Unoccupied AChR clusters are also found in the SMA animal at PND02 with snyaptophysin presynaptic staining. AChR clusters in the intercostal muscles of PND02 normal (A–C) carrier (D–F) and SMA (G–I) littermates. GFP and synaptophysin double staining reveals the motor neuron axons innervating the intercostal muscles in (A), (D), (G). Alpha-bungarotoxin labels the AChR clusters in (B), (E), (H), and merged confocal images are shown in (C), (F), (I). AChR clusters are fully innervated in normal and carrier animals while several AChR clusters are unoccupied in the SMA animal (arrowheads). In this micrograph a fully occupied AchR cluster is also shown (arrow). Scale bar represents 50 µm in each micrograph.

Figure 7.

Figure 7.

AChR cluster occupation in the SMA animal. GFP staining reveals the motor neuron axons innervating the intercostal muscles of e18.5 embryos (A, C, E) while alpha-bungarotoxin identifies the AChR clusters in the merged image (B, D, F). In some areas of the intercostal muscles at e18.5, the AChR clusters are fully occupied in both the carrier (A, B) and SMA animals (C, D). Additionally, often all AChR clusters in a particular area of the intercostal muscles are unoccupied in the SMA animal and little of the motor neuron axon remains (E, F). Scale bar represents 50 µm in each micrograph.

Figure 8.

Figure 8.

All AChR clusters are occupied in the diaphragm of SMA animals at PND03. AChR clusters in the diaphragm of PND03 normal (A–C) carrier (D–F) and SMA (G–I) littermates. GFP staining of motor axons (A, D, G), alpha-bungarotoxin staining of AChR clusters (B, E, H), Merged images (C, F, I). Scale bar: (A–I) 100 µm. Insert in images (C), (F), (I) represents 50 µm in width.

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