Research Article

Neuroscience

A novel mouse model for LAMA2-related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

Department of Pediatrics, Peking University First Hospital, China
Department of Neurology, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University, China
Institute of Cardiovascular Sciences and Key Laboratory of Molecular Cardiovascular Sciences, Peking University Health Science Center, China
Department of Urology, Sun Yat-sen University Cancer Center, China
Medical and Health Analysis Center, Peking University, China
New York State Institute for Basic Research in Developmental Disabilities, United States
Beijing Key Laboratory of Molecular Diagnosis and Study on Pediatric Genetic Diseases, China

Sep 17, 2025

https://doi.org/10.7554/eLife.94288.4

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8 figures and 6 additional files

Figures

Figure 1

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Generation of a mouse model with deletion of exon 3 (ΔExon 3) in the laminin alpha 2-chain gene (*Lama2*).

(A) Strategy of generation of ΔExon 3 mice (dy^H/dy^H mice) by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. The sequences of the *Lama2* gene marked with blue indicate sgRNAs targeting sequences, ones marked with brown indicate the location of polymerase chain reaction (PCR) primers (*Lama2*-F, *Lama2*-R1, and *Lama2*-R2), respectively. (B) PCR analysis for genotype identification of the F2 mice. (C) DNA sequencing of reverse transcription (RT)-PCR products from dy^H/dy^H muscle validated the deletion of exon 3, which resulted in a frameshift downstream sequence of *Lama2* gene.

Figure 1—source data 1 Original files for gels of polymerase chain reaction (PCR) analysis for genotype identification of the F2 mice by 2% agarose electrophoresis analysis displayed in Figure 1B.: https://cdn.elifesciences.org/articles/94288/elife-94288-fig1-data1-v1.zip
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Figure 1—source data 2 PDF file containing original gels of polymerase chain reaction (PCR) analysis for genotype identification for Figure 1B.: https://cdn.elifesciences.org/articles/94288/elife-94288-fig1-data2-v1.zip
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Figure 2 with 1 supplement

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General phenotype and muscle magnetic resonance imaging (MRI) of dy^H/dy^H mice.

(A) General phenotype of dy^H/dy^H mice. There was no significant difference in survival, body weight, muscle function (four-limb grip and the number of electric shocks on the treadmill), and serum creatine kinase (CK) levels between the wild-type (WT) and heterozygous mice at each point. Kaplan–Meier survival analysis revealed that the median survival of dy^H/dy^H mice (n=33) was 21 days (range 12–35 days), and none of the wild-type (n=18) and heterozygous (n=32) mice died during the observation period. Significant difference in body weight (g) between the WT and dy^H/dy^H mice was marked at P7 (3.74 vs 3.16, t-test, p<0.001) (WT: n=19; dy^H/dy^H: n=20), P10 (5.58 vs 4.09, t-test, p<0.001) (WT: n=11; dy^H/dy^H: n=16), P14 (6.81 vs 4.41, t-test, p<0.001) (WT: n=12; dy^H/dy^H: n=20), P17 (7.66 vs 5.04, t-test, p<0.001) (WT: n=12; dy^H/dy^H: n=14), P21 (8.34 vs 4.09, t-test, p<0.001) (WT: n=16; dy^H/dy^H: n=20), and P24 (10.41 vs 3.73, t-test, p<0.001) (WT: n=14; dy^H/dy^H: n=13). Two-week-old dy^H/dy^H mice could be easily identified due to their smaller size. Significant differences in the mean relative four-limb grip (force per gram body weight) between the WT and dy^H/dy^H mice were marked at P10 (3.16 vs 2.46, t-test, p<0.001) (WT: n=11; dy^H/dy^H: n=7), P14 (4.65 vs 3.37, t-test, p<0.001) (WT: n=18; dy^H/dy^H: n=17), P17 (5.05 vs 3.58, t-test, p=0.001) (WT: n=12; dy^H/dy^H: n=10), P21 (7.41 vs 5.12, t-test, p<0.001) (WT: n=12; dy^H/dy^H: n=9), and P24 (7.53 vs 4.73, t-test, p=0.003) (WT: n=11; dy^H/dy^H: n=3). The mean number of electric shocks on the treadmill significantly increased number of the electric shocks in dy^H/dy^H mice at P18 (t-test, p=0.010) (WT: n=13; dy^H/dy^H: n=9), P19 (t-test, p=0.012) (WT: n=13; dy^H/dy^H: n=8), P21 (t-test, p=0.003) (WT: n=13; dy^H/dy^H: n=6), P22 (t-test, p<0.001) (WT: n=13; dy^H/dy^H: n=6), P23 (t-test, p=0.017) (WT: n=13; dy^H/dy^H: n=5), and P24 (t-test, p=0.010) (WT: n=13; dy^H/dy^H: n=4). Approximately 9–10 times higher CK levels were detected in dy^H/dy^H mice than in WT mice at P14 (WT: n=8; dy^H/dy^H: n=7) and P21 (WT: n=8; dy^H/dy^H: n=6). *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, ns (p≥0.05), blue ‘ns’ for wild-type vs heterozygous mice, black ‘ns’ for vs dy^H/dy^H mice at two ages, and orange asterisks or ‘ns’ for WT vs dy^H/dy^H mice. (B) Muscle MRI in dy^H/dy^H mice. Pelvic and hindlimb muscle MRI showed significantly smaller muscle volumes on T1-weighted MRI and significantly increased hyperintense regions on T2-weighted muscle MRI in dy^H/dy^H mice (n=4) than in WT mice (n=4) at P14 and P21 (****p<0.001).

Figure 2—figure supplement 1

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Body weight and muscle function analysis of dy^H/dy^H, wild-type (WT), and Het mice.

Significant differences were shown in body weight, the mean relative four-limb grip (force per gram body weight), and the number of electric shocks on the treadmill between the WT and dy^H/dy^H mice at P21.

Figure 3 with 1 supplement

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Muscle pathology with age in the biceps femoris of dy^H/dy^H mice.

Hematoxylin and eosin (H&E) and Sirius Red staining of the biceps femoris were compared between wild-type and dy^H/dy^H mice at P1, P4, P7, P14, and P21. H&E staining showed a noticeable inequality in muscle fiber size in P7 dy^H/dy^H mice, the most extreme dystrophic changes (inequality in muscle fiber size, degeneration, necrosis, and regeneration of muscle fibers), and connective tissue penetration and inflammation in P14 dy^H/dy^H mice. Sirius Red staining showed increased collagen content and fibrosis in dy^H/dy^H mice from P7. Scale bars: 50 µm.

Figure 3—figure supplement 1

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Figure 4 with 1 supplement

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Extensive involvement with muscle pathology and laminin α2 deficiency in dy^H/dy^H mice.

(A) Hematoxylin and eosin (H&E) staining showed dystrophic changes in the quadriceps femoris, gastrocnemius, triceps brachii, diaphragm, and tongue muscles, but not as much as in the biceps femoris, while the heart and intestinal smooth muscles were spared. Scale bars: 50 µm. (B) The 300 kDa fragment of laminin α2 chain was detected in the biceps femoris of wild-type (WT) mice (n=3), but not in that of dy^H/dy^H mice (n=3) by Western blot analysis. (C) Colocalization of laminin α2 chain (green fluorescence, yellow arrow) and collagen VI (red fluorescence, blue arrow) showed that they were normally located and expressed in the basement membrane in WT muscle, but laminin α2 deficiency along with increased collagen VI were observed in dy^H/dy^H muscle.

Figure 4—source data 1 Original files for western blot analysis displayed in Figure 4B.: https://cdn.elifesciences.org/articles/94288/elife-94288-fig4-data1-v1.zip
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Figure 4—source data 2 PDF file containing original western blots for Figure 4B, indicating the relevant bands.: https://cdn.elifesciences.org/articles/94288/elife-94288-fig4-data2-v1.zip
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Figure 4—figure supplement 1

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Extensive involvement with muscle pathology in dy^H/dy^H mice at P21.

Hematoxylin and eosin (H&E) staining showed dystrophic changes in the quadriceps femoris, gastrocnemius, triceps brachii, diaphragm, and tongue muscles, but not as much as in the biceps femoris, while the heart and intestinal muscles were spared. Whole muscle, representative muscle area and magnified panels were shown. Scale bars: for the whole muscle: 200 µm; intermediate magnification panel: 50 µm, maximum magnification panel: 20 µm.

Figure 5 with 1 supplement

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Ssingle-cell RNA sequencing (scRNA-seq) analysis of brains from dy^H/dy^H and wild-type (WT) mice.

(A) UMAP visualization of all the cells colored by sample. (B) UMAP visualization of all the cells from dy^H/dy^H and WT brains colored by cluster identity. (C) Cell composition proportion of every annotated cell cluster in dy^H/dy^H and WT separately. (D) Violin plots of the expression of *Lama2* gene in WT and dy^H/dy^H in each annotated cell cluster. (E) Immunofluorescence of laminin α2, GFAP (astrocytes), and PLP1 on P14 mouse brain slices. DAPI was for nuclei staining. White arrows indicated the cortical surface. (F) Gene Ontology (GO) enrichment of differentially expressed genes in vascular and leptomeningeal fibroblasts, vascular smooth muscle cells, and astrocytes. (G) Cell-cell communications among cell clusters. Total number of inferred cell-cell interaction counts (left) and strength (right) in dy^H/dy^H and WT, respectively. Heatmap of ligand-receptor interaction counts in all pairwise cell clusters in WT and dy^H/dy^H brains.

Figure 5—figure supplement 1

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Single-cell RNA sequencing (scRNA-seq) analysis of dy^H/dy^H and wild-type (WT) mouse brains.

(A) Statistics of the sequencing results from each sample. (B) Violin plots show the number of UMI counts and detected genes in each sample. (**C, D**) Violin plots of the expression distribution of the selected markers for each cell cluster. The cell cluster annotation and the corresponding markers are as follows. neuron: Stmn2, Gap43, Snap25, Syp; hippocampal neuron (Hippo-neuron): Nhlh2, Reln; glutamatergic neuron (Glu-neuron): Slc17a6; GABAergic neuron (GABA-neuron): Gad2, Gad1, Slc32a1; neuron-glial antigen 2 neuron (NG2-neuron): Top2a, Mki67, Aspm; astrocyte (Aso): Agt, Fabp7, Gfap; cerebellum glia cell (CGC): Npy, Mybpc1; oligodendrocyte (Oligo): Plp1, Mbp, Mog; microglia (Micro): Cx3cr1; vascular and leptomeningeal fibroblasts (VLF): Slc6a13, Col1a2; vascular smooth muscle cell (VSM): Tagln, Acta2, Rgs5, Abcc9; choroid plexus cell (CPC): Folr1, Pcp4, Kl, Clic6; ependymal cell (Epen): Foxj1, Tmem212; endothelial cells (Endo): Fn1, Cldn5; macrophage (Macro): Mrc1. (E) UMAP visualization of *Lama2* expression in astrocyte clusters.

Figure 6

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Laminin pathway interactions and hypothesis for cortical dysplasia.

(A) The ligand-receptor pairs in laminin pathway. (B) Violin plots showed the expression of *Mbp*, *Mobp*, *Plp1*, *Slc1a2*, *Mt3*, and *Slc6a13* in every annotated cell cluster in dy^H/dy^H and wild-type (WT) separately. *** for p-value <0.001, ** for p-value <0.01, * for p-value <0.05. (C) Hypothesis that cortical dysplasia (cobblestone malformation) and neuronal over-migration in the cortex due to a defective gliovascular basal lamina of the blood-brain barrier caused by the lack of laminin α2 in *LAMA2*-MD.

Figure 7 with 1 supplement

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The transcriptional landscape of dy^H/dy^H (knockout, KO) mouse muscles in response to wild-type (WT) mouse muscles.

(A) Principal component analysis for dimension reduction of differentially expressed genes (DEGs) in the biceps femoris of dy^H/dy^H and dy^H/+mice in response to WT mice. (B) DEGs in dy^H/dy^H muscles relative to WT muscles were enriched in adhesion/extracellular matrix, cytoskeletal proteins and muscle development, ion channel and cell membrane transport, inflammation, mitochondrial metabolism, and apoptosis. (C) Protein-protein interactions between *Lama2* and integrins-related genes, including *Itgal*, *Itgax*, *Itgam*, *Itgb2*, and *Itgb7*. (D) The neuron death-related signaling pathways were enriched in dy^H/dy^H muscles relative to WT muscles by Gene Ontology (GO) pathway analyses.

Figure 7—figure supplement 1

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Immunofluorescence staining for α-dystroglycan in the biceps femoris of dy^H/dy^H mice.

Colocalization of α-dystroglycan (red fluorescence, blue arrow) and laminin α2 chain (green fluorescence, yellow arrow) showed that α-dystroglycan protein was normally located and expressed in the cell membrane both in the wild-type (WT) and dy^H/dy^H muscles. Scale bars: 40 µm.

Figure 8 with 2 supplements

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Changes of muscle cytoskeleton and development proteins in dy^H/dy^H mice.

(A) Immunofluorescence staining for myosin heavy chain (MYHC), desmin, β-tubulin, MYOG, MYOD1, and MYF-5 in WT and dy^H/dy^H muscles at P14. MYHC, MYOG, MYOD1, and MYF-5 were focally increased, while desmin and β-tubulin showed mild decrease in the cytoplasm of hypertrophic muscle fibers as well as focal increase in developmental muscle fibers in dy^H/dy^H mice. Wheat-germ agglutinin (WGA; red) was used to visualize the muscle fibers and connective tissue. White asterisk indicated focal regions with changes. (B) F-actin, α-actin, MYH2, MYHC, desmin, β-tubulin, MYOG, and MYOD1 were detected by Western blot in P14 WT (n=6) and dy^H/dy^H (n=6) muscles. The levels of F-actin, MYOG and MYOD1 were significantly decreased in dy^H/dy^H muscles (p<0.05), MYH2 and MYHC were significantly increased in dy^H/dy^H muscles (p<0.05), while α-actin, desmin, and β-tubulin showed no significant difference between WT (n=6) and dy^H/dy^H (n=6) mice.

Figure 8—source data 1 Original files for western blot analysis displayed in Figure 8B.: https://cdn.elifesciences.org/articles/94288/elife-94288-fig8-data1-v1.zip
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Figure 8—source data 2 PDF file containing original western blots for Figure 8B, indicating the relevant bands.: https://cdn.elifesciences.org/articles/94288/elife-94288-fig8-data2-v1.zip
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Figure 8—figure supplement 1

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Quantitative analysis of muscle cytoskeleton and development proteins in dy^H/dy^H mice.

(A) Quantitative analysis of immunofluorescence staining for myosin heavy chain (MYHC), MYOG, desmin, MYOD1, β-tubulin, and MYF-5 in wild-type (WT) and dy^H/dy^H muscles at P14. (B) Quantitative analysis of Western blot for laminin α2, F-actin, α-actin, MYH2, MYHC, desmin, β-tubulin, MYOG, and MYOD1 in P14 WT and dy^H/dy^H muscles. *** for p-value <0.001, ** for p-value <0.01, * for p-value <0.05.

Figure 8—figure supplement 2

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Proposed pathogenic mechanisms hypothesis in the dy^H/dy^H mouse model of laminin alpha 2-chain gene (*LAMA*2)-congenital muscular dystrophy (CMD).

Laminin α2 deficiency in the basement membrane due to pathogenic variants in the *LAMA2* gene leads to loss of dystroglycan/integrin-matrix scaffolds and extracellular matrix networks. Then, a series of molecular changes associated with the cytoskeleton, extracellular matrix, fibrosis, inflammation, apoptosis, pyroptosis, and mitochondrial energy metabolism occur.

Additional files

Supplementary file 1 Differentially expressed genes in all cell clusters in scRNA-seq analysis.: https://cdn.elifesciences.org/articles/94288/elife-94288-supp1-v1.xlsx
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Supplementary file 2 Differentially expressed genes (DEGs) (1136 upregulated and 884 downregulated) at least twofold (p<0.05) in dy^H/dy^H (KO) muscle samples relative to the wild-type (WT) muscle samples.: https://cdn.elifesciences.org/articles/94288/elife-94288-supp2-v1.xlsx
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Supplementary file 3 Comparison between the dy^H/dy^H mouse with other Lama2-deficient mice.: https://cdn.elifesciences.org/articles/94288/elife-94288-supp3-v1.docx
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Supplementary file 4 Primers of PCR amplifications for Genotype identification.: https://cdn.elifesciences.org/articles/94288/elife-94288-supp4-v1.docx
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Supplementary file 5 MRI examination imaging sequences.: https://cdn.elifesciences.org/articles/94288/elife-94288-supp5-v1.docx
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MDAR checklist: https://cdn.elifesciences.org/articles/94288/elife-94288-mdarchecklist1-v1.docx
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Dandan Tan
Yidan Liu
Huaxia Luo
Qiang Shen
Xingbo Long
Luzheng Xu
Jieyu Liu
Nanbert A Zhong
Hong Zhang
Hui Xiong

(2025)

A novel mouse model for LAMA2-related muscular dystrophy with analysis of molecular pathogenesis and clinical phenotype

eLife 13:RP94288.

https://doi.org/10.7554/eLife.94288.4

Figures

Generation of a mouse model with deletion of exon 3 (ΔExon 3) in the laminin alpha 2-chain gene (Lama2).

Figure 1—source data 1

Figure 1—source data 2

General phenotype and muscle magnetic resonance imaging (MRI) of dy^H/dy^H mice.

Body weight and muscle function analysis of dy^H/dy^H, wild-type (WT), and Het mice.

Muscle pathology with age in the biceps femoris of dy^H/dy^H mice.

Muscle pathology with age in the biceps femoris of dy^H/dy^H mice.

Extensive involvement with muscle pathology and laminin α2 deficiency in dy^H/dy^H mice.

Figure 4—source data 1

Figure 4—source data 2

Extensive involvement with muscle pathology in dy^H/dy^H mice at P21.

Ssingle-cell RNA sequencing (scRNA-seq) analysis of brains from dy^H/dy^H and wild-type (WT) mice.

Single-cell RNA sequencing (scRNA-seq) analysis of dy^H/dy^H and wild-type (WT) mouse brains.

Laminin pathway interactions and hypothesis for cortical dysplasia.

The transcriptional landscape of dy^H/dy^H (knockout, KO) mouse muscles in response to wild-type (WT) mouse muscles.

Immunofluorescence staining for α-dystroglycan in the biceps femoris of dy^H/dy^H mice.

Changes of muscle cytoskeleton and development proteins in dy^H/dy^H mice.

Figure 8—source data 1

Figure 8—source data 2

Quantitative analysis of muscle cytoskeleton and development proteins in dy^H/dy^H mice.

Proposed pathogenic mechanisms hypothesis in the dy^H/dy^H mouse model of laminin alpha 2-chain gene (LAMA2)-congenital muscular dystrophy (CMD).

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

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Generation of a mouse model with deletion of exon 3 (ΔExon 3) in the laminin alpha 2-chain gene (Lama2).

Figure 1—source data 1

Figure 1—source data 2

General phenotype and muscle magnetic resonance imaging (MRI) of dyH/dyH mice.

Body weight and muscle function analysis of dyH/dyH, wild-type (WT), and Het mice.

Muscle pathology with age in the biceps femoris of dyH/dyH mice.

Muscle pathology with age in the biceps femoris of dyH/dyH mice.

Extensive involvement with muscle pathology and laminin α2 deficiency in dyH/dyH mice.

Figure 4—source data 1

Figure 4—source data 2

Extensive involvement with muscle pathology in dyH/dyH mice at P21.

Ssingle-cell RNA sequencing (scRNA-seq) analysis of brains from dyH/dyH and wild-type (WT) mice.

Single-cell RNA sequencing (scRNA-seq) analysis of dyH/dyH and wild-type (WT) mouse brains.

Laminin pathway interactions and hypothesis for cortical dysplasia.

The transcriptional landscape of dyH/dyH (knockout, KO) mouse muscles in response to wild-type (WT) mouse muscles.

Immunofluorescence staining for α-dystroglycan in the biceps femoris of dyH/dyH mice.

Changes of muscle cytoskeleton and development proteins in dyH/dyH mice.

Figure 8—source data 1

Figure 8—source data 2

Quantitative analysis of muscle cytoskeleton and development proteins in dyH/dyH mice.

Proposed pathogenic mechanisms hypothesis in the dyH/dyH mouse model of laminin alpha 2-chain gene (LAMA2)-congenital muscular dystrophy (CMD).

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Supplementary file 5

MDAR checklist

Downloads (link to download the article as PDF)

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Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

General phenotype and muscle magnetic resonance imaging (MRI) of dy^H/dy^H mice.

Body weight and muscle function analysis of dy^H/dy^H, wild-type (WT), and Het mice.

Muscle pathology with age in the biceps femoris of dy^H/dy^H mice.

Muscle pathology with age in the biceps femoris of dy^H/dy^H mice.

Extensive involvement with muscle pathology and laminin α2 deficiency in dy^H/dy^H mice.

Extensive involvement with muscle pathology in dy^H/dy^H mice at P21.

Ssingle-cell RNA sequencing (scRNA-seq) analysis of brains from dy^H/dy^H and wild-type (WT) mice.

Single-cell RNA sequencing (scRNA-seq) analysis of dy^H/dy^H and wild-type (WT) mouse brains.

The transcriptional landscape of dy^H/dy^H (knockout, KO) mouse muscles in response to wild-type (WT) mouse muscles.

Immunofluorescence staining for α-dystroglycan in the biceps femoris of dy^H/dy^H mice.

Changes of muscle cytoskeleton and development proteins in dy^H/dy^H mice.

Quantitative analysis of muscle cytoskeleton and development proteins in dy^H/dy^H mice.

Proposed pathogenic mechanisms hypothesis in the dy^H/dy^H mouse model of laminin alpha 2-chain gene (LAMA2)-congenital muscular dystrophy (CMD).