Figures and data in Depletion of SMN protein in mesenchymal progenitors impairs the development of bone and neuromuscular junction in spinal muscular atrophy

Version of Record: February 6, 2024 Read the peer reviews
Reviewed Preprint: v2 January 23, 2024
Reviewed Preprint: v1 November 7, 2023

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Sang-Hyeon Hann

Seon-Yong Kim

Ye Lynne Kim

Young-Woo Jo

Jong-Seol Kang

Hyerim Park

Se-Young Choi

Young-Yun Kong

(2024)
Depletion of SMN protein in mesenchymal progenitors impairs the development of bone and neuromuscular junction in spinal muscular atrophy

eLife 12:RP92731.

https://doi.org/10.7554/eLife.92731.3
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Figures
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Additional files

8 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements

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Skeletal growth abnormalities and altered growth plate homeostasis in *SMN2* 1-copy *Smn1^ΔMPC* mice.

(A) Representative 3D images and longitudinal section view of the ossified femur bone. Scale bars, 1 mm. (**B, C**) *SMN2* 1-copy mutant’s femurs showed reduced growth in diaphysis length and diameter, …

Figure 1—figure supplement 1

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Growth defects in the *Prrx1*-lineage bone of *SMN2* 0-copy *Smn1^ΔMPC* mice.

(A) Representative control and mutant mice with 0 copies of *SMN2* were photographed at E18.5. (**B, C**) Alcian blue and alizarine red staining in the *SMN2* 0-copy *Smn1^ΔMPC* mutant showed abnormal skeletal …

Figure 1—figure supplement 2

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Osteoclasts and osteoblasts were undisturbed in *SMN2* 1-copy *Smn1^ΔMPC* mice.

(A) Cortical bone thicknesses were not significantly different between the control and mutant groups. (B) Bone mineral density was slightly increased in the diaphysis of *SMN2* 1-copy *Smn1^ΔMPC* mice. …

Figure 2

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Decreased chondrocyte-derived IGF–AKT axis by limb mesenchymal cell-specific survival motor neuron (SMN) depletion in *SMN2* 1-copy *Smn1^ΔMPC* mice.

(A) Representative images of p-AKT immunostaining in distal femur growth plate from mice at P14. Scale bars, 100 μm. (**B, C**) The p-AKT-positive percentage was decreased in the resting zone …

Figure 2—source data 1 Original file for the gel electrophoresis of genomic PCR in Figure 2G (Cre, Smn1^F7, Smn1^Δ7) and western blot analysis in Figure 2I (anti-alpha-tubulin, anti-SMN).: https://cdn.elifesciences.org/articles/92731/elife-92731-fig2-data1-v1.zip
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Figure 2—source data 2 PDF containing Figure 2G, I and original scans of the PCR and western blot with highlighted bands and sample labels.: https://cdn.elifesciences.org/articles/92731/elife-92731-fig2-data2-v1.zip
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Figure 3

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Aberrant postnatal neuromuscular junction (NMJ) maturation in *SMN2* 1-copy *Smn1^ΔMPC* mice.

(A) Immunostaining of NMJs in TA muscle of *SMN2* 1-copy *Smn1^WT* and *Smn1^ΔMPC* mice at P21 with anti-NF (green), anti-synaptophysin (blue), and α-Btx staining acetylcholine receptor (AChR; red). Scale …

Figure 4

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Morphological deterioration in neuromuscular junctions (NMJs) of adult *SMN2* 1-copy *Smn1^ΔMPC* mice.

(A) Immunostaining of NMJs in TA muscle of *SMN2* 1-copy *Smn1^WT*, *SMN2* 2-copy, and *SMN2* 1-copy *Smn1^ΔMPC* mice at P56 with anti-NF (green), anti-synaptophysin (blue), and α-Btx staining acetylcholine …

Figure 5

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Reduced presynaptic neurotransmission ability in the neuromuscular junctions (NMJs) of *SMN2* 1-copy *Smn1^ΔMPC* mice.

(A) Representative traces of Miniature endplate potential (mEPP) from *SMN2* 1-copy *Smn1*^ΔWT (top) and *SMN2* 1-copy *Smn1^ΔMPC* (bottom) mice. (**B, C**) *SMN2* 1-copy mutant’s NMJs showed an increase in mEPP …

Figure 6

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Abnormal nerve terminal ultrastructure in *SMN2* 1-copy mutant.

(A) Representative transmission electron microscopy (TEM) images from neuromuscular junctions (NMJs) of *SMN2* 1-copy *Smn1*^ΔWT and *SMN2* 1-copy *Smn1^ΔMPC* mice at P56. Scale bars, 500 nm. Nerve terminal …

Figure 7

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Improved postnatal neuromuscular junction (NMJ) development in the TA muscle of *SMN2* 1-copy *Smn1^ΔMPC* mice following healthy fibro-adipogenic progenitors (FAPs) transplantation.

(A) Immunostaining of NMJs in tdTomato⁺ FAP-transplanted TA muscle (+FAP) and vehicle-treated contralateral muscle (+Veh) in *SMN2* 1-copy *Smn1^ΔMPC* mice at P56 with anti-NF (yellow), …

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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Mus musculus)	Prrx1^Cre	The Jackson Laboratory	Strain #: 005584; RRID:IMSR_JAX:005584
Genetic reagent (M. musculus)	Smn1^f7/+	The Jackson Laboratory	Strain #: 006138; RRID:IMSR_JAX:006138
Genetic reagent (M. musculus)	Rosa26^LSL-YFP/+	The Jackson Laboratory	Strain #: 006148; RRID:IMSR_JAX:006148
Genetic reagent (M. musculus)	Rosa26^{LSL-tdTomato/+}	The Jackson Laboratory	Strain #: 007914; RRID:IMSR_JAX:007914
Genetic reagent (M. musculus)	SMN2^+/+; Smn1^+/−	The Jackson Laboratory	Strain #: 005024; RRID:IMSR_JAX:005024
Genetic reagent (M. musculus)	Smn1^+/−; SMN2^+/+; SMNΔ7^+/+	The Jackson Laboratory	Strain #: 005025; RRID:IMSR_JAX:005025
Antibody	anti-SMN (Mouse monoclonal)	BD Biosciences	Cat. #: 610646; RRID:AB_397973	WB (1:1000)
Antibody	anti-alpha-tubulin (Rabbit monoclonal)	Abcam	Cat. #: ab176560; RRID:AB_2860019	WB (1:1000)
Antibody	anti-Neurofilament M (Rabbit polyclonal)	Merckmillipore	Cat. #: ab1987; RRID:AB_91201	IF (1:1000)
Antibody	anti-Synaptophysin 1 (Guinea pig polyclonal)	Synaptic Systems	Cat. #: 101 004; RRID:AB_1210382	IF (1:500)
Antibody	anti-GFP (Chicken polyclonal)	Abcam	Cat. #: ab13970; RRID:AB_300798	IF (1:500)
Antibody	anti-Ki67 (Rabbit polyclonal)	Abcam	Cat. #: ab15580; RRID:AB_443209	IF (1:500)
Antibody	anti-Itgb3 (Rabbit monoclonal)	Cell Signaling	Cat. #: 13166; RRID:AB_2798136	IF (1:100)
Antibody	anti-p-AKT(S473) (Rabbit monoclonal)	Cell Signaling	Cat. #: 4060; RRID:AB_2315049	IF (1:100)
Antibody	anti-CD45-APC (Rat monoclonal)	Biolegend	Cat. #: 103111; RRID:AB_312976	FACS (3 µl per test)
Antibody	anti-CD31-APC (Rat monoclonal)	Biolegend	Cat. #: 102409; RRID:AB_312904	FACS (3 µl per test)
Antibody	anti-Sca-1(Ly6a)-FITC (Rat monoclonal)	Biolegend	Cat. #: 122507; RRID:AB_756192	FACS (3 µl per test)
Antibody	anti-Sca-1(Ly6a)-Pacific blue (Rat monoclonal)	Biolegend	Cat. #: 108120; RRID:AB_493273	FACS (3 µl per test)
Antibody	anti-Vcam1-Biotin (Rat monoclonal)	Biolegend	Cat. #: 105703; RRID:AB_313204	FACS (3 µl per test)
Antibody	anti-Rabbit IgG-HRP (Goat monoclonal)	Promega	Cat. #: W4011; RRID:AB_430833	WB (1:10,000)
Antibody	anti-Mouse IgG-HRP (Goat monoclonal)	Promega	Cat. #: W4021; RRID:AB_430834	WB (1:10,000)
Antibody	anti-Rabbit IgG-Alexa fluor 488 (Goat monoclonal)	Invitrogen	Cat. #: A11034; RRID:AB_2576217	IF (1:500)
Antibody	anti-Chicken IgY-Alexa fluor 488 (Goat monoclonal)	Invitrogen	Cat. #: A11039; RRID:AB_2534096	IF (1:500)
Antibody	anti-Rabbit IgG-Alexa fluor Plus 647 (Goat monoclonal)	Invitrogen	Cat. #: A32733; RRID:AB_2633282	IF (1:500)
Antibody	anti-Guine pig IgG-Alexa fluor 405 (Goat monoclonal)	Abcam	Cat. #: ab175678; RRID:AB_2827755	IF (1:500)
Peptide, recombinant protein	PE-Cy7-Streptavidin	Biolegend	Cat. #: 405206	FACS (3 µl per test)
Peptide, recombinant protein	Alpha-bungarotoxin-Alexa fluor 555	Invitrogen	Cat. #: B35451	IF (1:1000)
Peptide, recombinant protein	Alpha-bungarotoxin-Alexa fluor 488	Invitrogen	Cat. #: B13422	IF (1:1000)
Peptide, recombinant protein	μ-conotoxin GIIIB	Alomone	Cat. #: C-270	Electrophysiology (2.5 µM)
Commercial assay or kit	Pierce BCA protein assay kits	Thermo Fisher Scientific	Cat. #: C-23225
Software	ImageJ	ImageJ	RRID:SCR_003070
Software	AccuCT	PerkinElmer
Software	Microsoft Excel	Microsoft	RRID:SCR_016137
Software	Leica Application Suite X	Leica	RRID:SCR_013673
Software	GraphPad Prism	Graphpad	RRID:SCR_002798
Software	pClamp	Molecular devices	RRID:SCR_011323
Software	QIAGEN	QIAGEN	RRID:SCR_008539
Other	Gill No. 3 formula hematoxylin	Sigma	Cat. #: GHS332	Hematoxylin staining solution (1×)
Other	Eosin Y solution	Sigma	Cat. #: HT110116	Eosin staining solution (1×)
Other	Toluidine blue	Sigma	Cat. #: 198161	Toluidine blue staining (1%)

Additional files

MDAR checklist: https://cdn.elifesciences.org/articles/92731/elife-92731-mdarchecklist1-v1.docx
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Supplementary file 1 A primer list of genomic PCR and qRT-PCR.: https://cdn.elifesciences.org/articles/92731/elife-92731-supp1-v1.xlsx
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Figures

Skeletal growth abnormalities and altered growth plate homeostasis in SMN2 1-copy Smn1^ΔMPC mice.

Growth defects in the Prrx1-lineage bone of SMN2 0-copy Smn1^ΔMPC mice.

Osteoclasts and osteoblasts were undisturbed in SMN2 1-copy Smn1^ΔMPC mice.

Decreased chondrocyte-derived IGF–AKT axis by limb mesenchymal cell-specific survival motor neuron (SMN) depletion in SMN2 1-copy Smn1^ΔMPC mice.

Figure 2—source data 1

Figure 2—source data 2

Aberrant postnatal neuromuscular junction (NMJ) maturation in SMN2 1-copy Smn1^ΔMPC mice.

Morphological deterioration in neuromuscular junctions (NMJs) of adult SMN2 1-copy Smn1^ΔMPC mice.

Reduced presynaptic neurotransmission ability in the neuromuscular junctions (NMJs) of SMN2 1-copy Smn1^ΔMPC mice.

Abnormal nerve terminal ultrastructure in SMN2 1-copy mutant.

Improved postnatal neuromuscular junction (NMJ) development in the TA muscle of SMN2 1-copy Smn1^ΔMPC mice following healthy fibro-adipogenic progenitors (FAPs) transplantation.

Tomato+ FAPs in muscle.

Tables

Additional files

MDAR checklist

Supplementary file 1

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Skeletal growth abnormalities and altered growth plate homeostasis in SMN2 1-copy Smn1ΔMPC mice.

Growth defects in the Prrx1-lineage bone of SMN2 0-copy Smn1ΔMPC mice.

Osteoclasts and osteoblasts were undisturbed in SMN2 1-copy Smn1ΔMPC mice.

Decreased chondrocyte-derived IGF–AKT axis by limb mesenchymal cell-specific survival motor neuron (SMN) depletion in SMN2 1-copy Smn1ΔMPC mice.

Figure 2—source data 1

Figure 2—source data 2

Aberrant postnatal neuromuscular junction (NMJ) maturation in SMN2 1-copy Smn1ΔMPC mice.

Morphological deterioration in neuromuscular junctions (NMJs) of adult SMN2 1-copy Smn1ΔMPC mice.

Reduced presynaptic neurotransmission ability in the neuromuscular junctions (NMJs) of SMN2 1-copy Smn1ΔMPC mice.

Abnormal nerve terminal ultrastructure in SMN2 1-copy mutant.

Improved postnatal neuromuscular junction (NMJ) development in the TA muscle of SMN2 1-copy Smn1ΔMPC mice following healthy fibro-adipogenic progenitors (FAPs) transplantation.

Tomato+ FAPs in muscle.

MDAR checklist

Supplementary file 1

Skeletal growth abnormalities and altered growth plate homeostasis in SMN2 1-copy Smn1^ΔMPC mice.

Growth defects in the Prrx1-lineage bone of SMN2 0-copy Smn1^ΔMPC mice.

Osteoclasts and osteoblasts were undisturbed in SMN2 1-copy Smn1^ΔMPC mice.

Decreased chondrocyte-derived IGF–AKT axis by limb mesenchymal cell-specific survival motor neuron (SMN) depletion in SMN2 1-copy Smn1^ΔMPC mice.

Aberrant postnatal neuromuscular junction (NMJ) maturation in SMN2 1-copy Smn1^ΔMPC mice.

Morphological deterioration in neuromuscular junctions (NMJs) of adult SMN2 1-copy Smn1^ΔMPC mice.

Reduced presynaptic neurotransmission ability in the neuromuscular junctions (NMJs) of SMN2 1-copy Smn1^ΔMPC mice.

Improved postnatal neuromuscular junction (NMJ) development in the TA muscle of SMN2 1-copy Smn1^ΔMPC mice following healthy fibro-adipogenic progenitors (FAPs) transplantation.