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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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Muscle development is normal when Smn is decreased. Nomarski lateral views of mid-trunk muscle from 50-h gata2–GFP transgenic embryos injected with 9 ng of control MO (A; n = 20) or smn MO (B; n = 20) showing somitic boundaries (black arrows). Dorsal views of 22-h whole-mount in situ hybridization of myoD (purple; black arrowhead) in control MO–injected (C; n = 31) and smn MO–injected (D; n = 18) embryos. Cross section of 27-h znp1 (motor axon; arrows) and F59 (slow muscle; arrowheads) mAb–stained embryos injected with control MO (E; n = 40) or smn MO (F; n = 65). Cross section of 27-h znp1 (motor axon; arrows) and F310 (fast muscle; arrowheads) mAb–stained embryos injected with control MO (G; n = 20) or smn MO (H; n = 41). Bars: (A–D) 75 μm; (E–H) 30 μm.
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fig7: Muscle development is normal when Smn is decreased. Nomarski lateral views of mid-trunk muscle from 50-h gata2–GFP transgenic embryos injected with 9 ng of control MO (A; n = 20) or smn MO (B; n = 20) showing somitic boundaries (black arrows). Dorsal views of 22-h whole-mount in situ hybridization of myoD (purple; black arrowhead) in control MO–injected (C; n = 31) and smn MO–injected (D; n = 18) embryos. Cross section of 27-h znp1 (motor axon; arrows) and F59 (slow muscle; arrowheads) mAb–stained embryos injected with control MO (E; n = 40) or smn MO (F; n = 65). Cross section of 27-h znp1 (motor axon; arrows) and F310 (fast muscle; arrowheads) mAb–stained embryos injected with control MO (G; n = 20) or smn MO (H; n = 41). Bars: (A–D) 75 μm; (E–H) 30 μm.

Mentions: To determine whether the motor axon defects observed were due to defects in muscle development, we examined the development and patterning of the myotomes. Overall myotome morphology was analyzed using live gata2–GFP transgenic embryos injected with either control MO or smn MO. Nomarski images were taken of muscle segments containing defective GFP-expressing nerves. No difference was seen in the muscle morphology between control MO– and smn MO–injected embryos (Fig. 7, A and B). No defects were seen in early muscle patterning in smn MO–injected embryos compared with control MO, as assayed by myoD expression, a gene expressed in the posterior region of the developing somite (Weinberg et al., 1996; Fig. 7, C and D). Lastly, we asked whether muscle cells were correctly specified by characterizing slow and fast muscle in smn MO–injected embryos. Using the slow muscle mAb F59 (Crow and Stockdale, 1986; Devoto et al., 1996) and the fast muscle mAb F310 (Crow and Stockdale, 1986; Zeller et al., 2002), we found no difference between muscle specification and organization upon depletion of Smn (Fig. 7, E–H). These data suggest that low levels of Smn do not adversely affect muscle organization, specification, or 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)

Muscle development is normal when Smn is decreased. Nomarski lateral views of mid-trunk muscle from 50-h gata2–GFP transgenic embryos injected with 9 ng of control MO (A; n = 20) or smn MO (B; n = 20) showing somitic boundaries (black arrows). Dorsal views of 22-h whole-mount in situ hybridization of myoD (purple; black arrowhead) in control MO–injected (C; n = 31) and smn MO–injected (D; n = 18) embryos. Cross section of 27-h znp1 (motor axon; arrows) and F59 (slow muscle; arrowheads) mAb–stained embryos injected with control MO (E; n = 40) or smn MO (F; n = 65). Cross section of 27-h znp1 (motor axon; arrows) and F310 (fast muscle; arrowheads) mAb–stained embryos injected with control MO (G; n = 20) or smn MO (H; n = 41). Bars: (A–D) 75 μm; (E–H) 30 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1761110&req=5

fig7: Muscle development is normal when Smn is decreased. Nomarski lateral views of mid-trunk muscle from 50-h gata2–GFP transgenic embryos injected with 9 ng of control MO (A; n = 20) or smn MO (B; n = 20) showing somitic boundaries (black arrows). Dorsal views of 22-h whole-mount in situ hybridization of myoD (purple; black arrowhead) in control MO–injected (C; n = 31) and smn MO–injected (D; n = 18) embryos. Cross section of 27-h znp1 (motor axon; arrows) and F59 (slow muscle; arrowheads) mAb–stained embryos injected with control MO (E; n = 40) or smn MO (F; n = 65). Cross section of 27-h znp1 (motor axon; arrows) and F310 (fast muscle; arrowheads) mAb–stained embryos injected with control MO (G; n = 20) or smn MO (H; n = 41). Bars: (A–D) 75 μm; (E–H) 30 μm.
Mentions: To determine whether the motor axon defects observed were due to defects in muscle development, we examined the development and patterning of the myotomes. Overall myotome morphology was analyzed using live gata2–GFP transgenic embryos injected with either control MO or smn MO. Nomarski images were taken of muscle segments containing defective GFP-expressing nerves. No difference was seen in the muscle morphology between control MO– and smn MO–injected embryos (Fig. 7, A and B). No defects were seen in early muscle patterning in smn MO–injected embryos compared with control MO, as assayed by myoD expression, a gene expressed in the posterior region of the developing somite (Weinberg et al., 1996; Fig. 7, C and D). Lastly, we asked whether muscle cells were correctly specified by characterizing slow and fast muscle in smn MO–injected embryos. Using the slow muscle mAb F59 (Crow and Stockdale, 1986; Devoto et al., 1996) and the fast muscle mAb F310 (Crow and Stockdale, 1986; Zeller et al., 2002), we found no difference between muscle specification and organization upon depletion of Smn (Fig. 7, E–H). These data suggest that low levels of Smn do not adversely affect muscle organization, specification, or 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