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Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding.

McWhorter ML, Monani UR, Burghes AH, Beattie CE - J. Cell Biol. (2003)

Bottom Line: As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development.Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously.These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Neurobiology, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by a loss of alpha motoneurons in the spinal cord. SMA is caused by low levels of the ubiquitously expressed survival motor neuron (Smn) protein. As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development. Using antisense morpholinos to reduce Smn levels throughout the entire embryo, we found motor axon-specific pathfinding defects. Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously. These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.

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Related in: MedlinePlus

Motor axons/nerves are abnormal in embryos injected with 9 ng of smn MO. Western blot analysis of WT uninjected (lanes 1 and 3), control MO–injected (lane 2), and smn MO–injected (9 ng) (lane 4) embryos at 36 h. Hu-C, a neuronal marker, is shown as a loading control. Lateral views of whole-mount embryos labeled with znp1 mAb at 27 (C–E) and 36 h (F–H) in embryos injected with control MO (C and F) or smn MO (D, E, G, and H). Truncated motor axons/nerves (D and G; black arrowheads) and branched motor axons/nerves (E and H; black arrows) occur when Smn protein levels are further reduced. Bars: (C–E) 25 μm; (F–H) 30 μm.
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fig2: Motor axons/nerves are abnormal in embryos injected with 9 ng of smn MO. Western blot analysis of WT uninjected (lanes 1 and 3), control MO–injected (lane 2), and smn MO–injected (9 ng) (lane 4) embryos at 36 h. Hu-C, a neuronal marker, is shown as a loading control. Lateral views of whole-mount embryos labeled with znp1 mAb at 27 (C–E) and 36 h (F–H) in embryos injected with control MO (C and F) or smn MO (D, E, G, and H). Truncated motor axons/nerves (D and G; black arrowheads) and branched motor axons/nerves (E and H; black arrows) occur when Smn protein levels are further reduced. Bars: (C–E) 25 μm; (F–H) 30 μm.

Mentions: 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.


Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding.

McWhorter ML, Monani UR, Burghes AH, Beattie CE - J. Cell Biol. (2003)

Motor axons/nerves are abnormal in embryos injected with 9 ng of smn MO. Western blot analysis of WT uninjected (lanes 1 and 3), control MO–injected (lane 2), and smn MO–injected (9 ng) (lane 4) embryos at 36 h. Hu-C, a neuronal marker, is shown as a loading control. Lateral views of whole-mount embryos labeled with znp1 mAb at 27 (C–E) and 36 h (F–H) in embryos injected with control MO (C and F) or smn MO (D, E, G, and H). Truncated motor axons/nerves (D and G; black arrowheads) and branched motor axons/nerves (E and H; black arrows) occur when Smn protein levels are further reduced. Bars: (C–E) 25 μm; (F–H) 30 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1761110&req=5

fig2: Motor axons/nerves are abnormal in embryos injected with 9 ng of smn MO. Western blot analysis of WT uninjected (lanes 1 and 3), control MO–injected (lane 2), and smn MO–injected (9 ng) (lane 4) embryos at 36 h. Hu-C, a neuronal marker, is shown as a loading control. Lateral views of whole-mount embryos labeled with znp1 mAb at 27 (C–E) and 36 h (F–H) in embryos injected with control MO (C and F) or smn MO (D, E, G, and H). Truncated motor axons/nerves (D and G; black arrowheads) and branched motor axons/nerves (E and H; black arrows) occur when Smn protein levels are further reduced. Bars: (C–E) 25 μm; (F–H) 30 μm.
Mentions: 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.

Bottom Line: As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development.Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously.These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Neurobiology, The Ohio State University, Columbus, OH 43210, USA.

ABSTRACT
Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by a loss of alpha motoneurons in the spinal cord. SMA is caused by low levels of the ubiquitously expressed survival motor neuron (Smn) protein. As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development. Using antisense morpholinos to reduce Smn levels throughout the entire embryo, we found motor axon-specific pathfinding defects. Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously. These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.

Show MeSH
Related in: MedlinePlus