Table 1.
Primers sequences and amplicon size used in qRT-PCR analyses.
Fig 1.
Gross fetal morphology at E14.5 and E18.5.
Photographs of whole fetuses were obtained at E14.5 and E18.5 for RYR1+/+ (WT), RYR1+/- and RYR1-/- littermates (A), as well as for Cav1.1+/+ (WT), Cav1.1+/- and Cav1.1-/- littermates (B).
Fig 2.
Histology of mouse limb skeletal muscle at embryonic day E14.5.
Cross sections of the lower hind limb of a WT fetus (A-C), a RYR1+/- fetus (D-F), a RYR1-/- fetus (G-I), a Cav1.1+/- fetus (J-L), and a Cav1.1-/- fetus (M-O), respectively. At E14.5, the skeletal muscle of the hind limb of a WT fetus (A, B) as well as of a RYR1+/- fetus (D, E) already harbor muscle fascicles (surrounded by dotted line) consisting of numerous muscle fibers while myoblasts were virtually absent. In contrast, the skeletal muscle of the hind limb of a RYR1-/- (G, H), a Cav1.1+/- (J, K), and a Cav1.1-/- (M, N) fetus, respectively, exhibits disorganization (asterisks) or complete absence of muscle fascicles and numerous myoblasts. Immunohistochemistry with anti-activated caspase-3 reveals prominent apoptosis only in nuclei of the myotubes of a Cav1.1-/- fetus at E14.5 (O, arrows). H&E staining (A, B, D, E, G, H, J, K, M, and N); original magnification x100 (A, D, G, J, M) and x200 (B, E, H, K, N). Immunohistochemistry with rabbit anti-mouse activated caspase-3 (clone C92-605; BD Biosciences) and slight counterstaining with hemalum; original magnification x400. Scale bars correspond to 100 μm in all microphotographs.
Fig 3.
Histology of mouse limb skeletal muscle at embryonic day E18.5.
Cross sections of the lower hind limb of a WT fetus (A-C), a RYR1+/- fetus (D-F), a RYR1-/- fetus (G-I), a Cav1.1+/- fetus (J-L), and a Cav1.1-/- fetus (M-O), respectively. At E18.5, the fetal skeletal muscles of the various genetically modified mice exhibit more pronounced morphological alterations. At this time point, the skeletal muscle of a WT fetus (A-C) is mature with regularly developed muscle fascicles consisting of normal sized muscle fibers as well as inconspicuous bone having reached a normal state of mineralization. In a RYR1+/- fetus (D-F), skeletal muscle and bone are normally developed, thus, being similar to WT mice. In contrast, the skeletal muscle of a RYR1-/- (G,H) and a Cav1.1-/- (M,N) fetus, respectively, consists predominantly of small, unorganized myotubes with lack of a fascicular organization. In addition, bone of the hind limb of a Cav1.1-/- (M-O) fetus is impaired in development as evidenced by persisting hyaline cartilage while mineralization has not been initiated (arrows in M). At day E18.5 apoptosis is completely absent from all mutant strains as evidenced by the absence of nuclear immunoreaction in immunohistochemistry with anti-activated caspase-3. H&E staining (A, B, D, E, G, H, J, K, M, and N); original magnification x100 (A, D, G, J, M) and x200 (B, E, H, K, N). Immunohistochemistry with rabbit anti-mouse activated caspase-3 (clone C92-605; BD Biosciences) and slight counterstaining with hemalum; original magnification x400. Scale bars correspond to 100 μm in all microphotographs.
Fig 4.
Comparison of the expression of MRFs in skeletal muscle from WT, RYR1-/- and Cav1.1-/- mice at E14.5 and at E18.5.
Relative expression levels of Six1, Six4, Pax3, Pax7, Myf5, Myod1, Myog and Mrf4 in WT, RYR1-/- and Cav1.1-/- samples (each n = 6) at E14.5 (upper part) and E18.5 (lower part) were obtained by qRT-PCR analyses, using Cytb as endogenous control. Expression levels of WT samples were set to 1. One way ANOVA with Bonferroni’s Multiple Comparison tests were performed for each gene, *represents a p-value ≤ 0.05. Error bars are S.E.M.
Fig 5.
Cav1.1 splice variants in WT and RYR1-/- skeletal muscle.
(A) Graphical representation of the genomic exon 29 vicinity of murine full-length and Δ29 Cav1.1 (NCBI Reference Sequence: NM_001081023.1) splice variants. Arrows indicate the primer binding positions used for amplification of exons 28–32. (B) PCR products of the full-length (343 bp) and Δ29 (286 bp) Cav1.1 splice variants. (C) Full-length (343 bp) and Δ29 (286 bp) splice variants as percentage of total Cav1.1 mRNA in limb skeletal muscle from WT and RYR1-/- animals at E14.5 and E18.5. (D) Relative expression of total Cav1.1 mRNA measured via qRT-PCR in RYR1-/- vs. WT skeletal muscle at E14.5 and E18.5, using Cytb as endogenous control. t-tests were performed for comparison of Δ29 vs. full-length splice variants (C) and for WT vs. RYR1-/- (D) in each group; * indicates p values <0.05 and *** p values < 0.001; error bars are S.E.M.
Fig 6.
Schematic representation of the samples used in the present study and work flow for the subsequent gene expression analyses.
Heterozygous Cav1.1+/- and RYR1+/- male and female animals were subjected to timed pairings. At E14.5 and E18.5 post coitum three pregnant females of each line were sacrificed and skeletal muscle samples were collected from the front and hind limbs of 3 littermates (n = 3) of each of the genotypes—WT, heterozygous (Cav1.1+/- or RYR1+/-) and homozygous (Cav1.1-/- or RYR1-/-) mutants. Samples were handled separately and used for total RNA extractions and subsequent MA and qRT-PCR analyses.
Fig 7.
Initial MAs and DEGs analysis.
(A) A principal component analysis (PCA) was performed for all samples with all their genes detected by the MAs via the Transcriptome Analysis Console 3.0 (Affymetrix®). (B) When comparing different developmental stages (E18.5 vs. E14.5), the cut-off criteria for being considered as DEG were an FC ≥ +2 or ≤-2, and a p ≤ 0.05 (the example shown is from the comparison WT E18.5 vs. WT E14.5). (C) When comparing groups from the same developmental stage, the cut-off criteria were an FC ≥ +1.5 or ≤-1.5, and a p ≤ 0.05 (the example shown is from the comparison Cav1.1-/- E18.5 vs. WT E18.5).
Table 2.
Differentially regulated genes for various comparisons of genotypes.
Fig 8.
Validation of the MAs results via qRT-PCRs.
(A-C) Validation of DEGs found in the comparison E18.5 vs. E14.5 for the same genotype. (A), WT vs. WT, 8 genes, n = 6 biological replicates per group; (B), RYR1-/- vs. RYR1-/-, 7 genes, n = 3 biological replicates per group; (C), Cav1.1-/- vs. Cav1.1-/-, 7 genes, n = 3 biological replicates per group. (D-E) Validation of selected genes found to be differentially regulated in E14.5 samples from RYR1-/- muscle (D) and Cav1.1-/- muscle (E), when compared to E14.5 WT. (D & E), 4 genes for each genotype comparison, n = 3 biological replicates per group. (F-G) Validation of selected genes found to be differentially regulated in E18.5 samples from RYR1-/- muscle (F) and Cav1.1-/- muscle (G), when compared to E18.5 WT. (F), 6 genes, n = 3 biological replicates per group; (G), 7 genes, n = 3 biological replicates per group. In all MA and qRT-PCR analyses the FCs of the control samples were set to 1. The relative expression levels obtained by qRT-PCR analysis were normalized to Cytb, which was used as endogenous control. Error bars are S.E.M.
Fig 9.
Biological processes affected by the RYR1-/- and Cav1.1-/- mutations at E14.5.
GO BP enrichment analyses were performed for the DEGs identified in the RYR1-/- (A) and Cav1.1-/- (B) samples when compared to WT littermates samples at E14.5. The ten most significantly enriched categories for each analysis are shown. Arrows indicate categories presented as heat maps in (C) and (D). The enrichment analyses was performed via the Enrichr online tool [31], length of the bars represents the significance (p-value). Heatmaps were generated for the DEGs enriched in the “Regulation of neuron differentiation” biological process in RYR1-/- samples (C) and for the DEGs enriched in “Muscle contraction” biological process in Cav1.1-/- samples (D). The heatmaps were generated from the MAs intensity levels of each gene via ClustVis [32]. Hierarchical average linkage clustering using the Euclidean distance was performed for all rows and columns.
Fig 10.
Biological processes affected by the RYR1-/- and Cav1.1-/- mutations at E18.5.
GO BP enrichment analyses were performed for the DEGs identified in the RYR1-/- (A) and Cav1.1-/- (B) samples with their E18.5 WT littermates serving as reference. The ten most significantly enriched categories for each analysis are shown. Arrows indicate categories presented as heat maps in (C), (D) and (E). The enrichment analyses was performed via the Enrichr online tool [31], the length of the bars corresponds to the significance (p-value). Heatmaps were generated for the DEGs enriched in the “Muscle contraction” biological process in RYR1-/- and Cav1.1-/- samples (C); for the DEGs enriched in “Extracellular matrix organization” biological process in RYR1-/- samples (D); and for the DEGs enriched in “Acylglycerol metabolic process” biological process in Cav1.1-/- samples (E). Heatmaps were generated from the MAs intensity levels of each included gene via ClustVis [32]. Hierarchical average linkage clustering using the Euclidean distance was performed for all rows and columns.
Fig 11.
Analysis of all DEGs found in skeletal muscle development from E14.5 to E18.5.
GO BP (A, C and E) and Wiki Pathways (B, D, F) enrichment analyses of all DEGs found in WT (A, B), RYR1-/- (C, D) and Cav1.1-/- (E,F) from E14.5 (control) to E18.5. The ten most significantly enriched categories for each analysis are shown. Enrichment analyses (A–F) were performed via the Enrichr online tool [31], length of the bars is proportional to the significance (p-value).
Fig 12.
DEGs specific for the E14.5 to E18.5 development of WT, RYR1-/- or Cav1.1-/- skeletal muscle.
(A) A Venn diagram, showing the number of DEGs identified in the MA analyses at E18.5 compared to E14.5 in WT, RYR1-/- and Cav1.1-/- limb skeletal muscle. Numbers in the overlapping and non-overlapping areas represent the amount of shared and not shared DEGs between genotypes, respectively. Wiki Pathways (B, C, D), GO BP (E, G, I) and GO CC (F, H, J) enrichment analyses of the DEGs found exclusively in WT (483 DEGs, blue charts), RYR1-/- (91 DEGs, yellow charts) and Cav1.1-/- (171 DEGs, red charts) from E14.5 (control) to E18.5, respectively. The ten most significantly enriched categories for each analysis are shown. Enrichment analyses (B–J) were performed via the Enrichr online tool [31], length of the bars is proportional to the significance (p-value). Gray bars in (J) indicate a p-value ≥ 0.05.
Fig 13.
miRNAs identified by the MAs during WT skeletal muscle development.
(A) Up (orange) and down (blue) regulated DEGs identified by the MAs for E18.5 vs. E14.5 (E14.5 is control) taking part in the Wiki pathway “Mirs in Muscle Cell Differentiation Homo sapiens (2012)”. DEGs regulated only in WT samples from E14.5 to E18.5 are shown in bold. (B) A heat map of all miRNAs, found to be differentially regulated at E18.5 compared to E14.5 in WT samples. Each row represents one biological replicate. miRNAs, found to be differentially regulated from E14.5 to E18.5 also in RYR1-/- samples, are underlined in yellow, in the Cav1.1-/- samples in red, and in both RYR1-/- and Cav1.1-/- samples in yellow and red, respectively. The heatmap was generated from the MAs intensity levels of each of the Mir genes via ClustVis [32].
Table 3.
miRNAs differentially regulated from E14.5 to E18.5 in WT, RYR1-/- and Cav1.1-/-.