Hindlimb Muscles of 17.5–18.5 dpc Mice Double Null for MyoD and Trp53 Appear Indistinguishable from Muscles of Mice Null for Either Gene
Authors: Im Chi Hyon , Chau Kevin , Lopez Mark , Sun Xiu Zhu , Tran John , Cuya Selma , Sharp Sandra B 
Published: November 2023

It has been shown by others that mice with either a MyoD null (MN) or Trp53 null (PN) genotype undergo myogenesis resulting in perinatal hindlimb muscle essentially indistinguishable from that of wild type (WT), while myogenic cells of the same null genotypes undergo impaired differentiation in culture. Both MyoD and Trp53 act through p21 and pRb to influence cell cycle withdrawal and differentiation. We hypothesized that redundancies in the activities of Trp53 and MyoD might rescue in vivo myogenesis in animals null for either protein, in which case animals double null (DN) for MyoD and Trp53 should demonstrate impaired hindlimb myogenesis. We bred PN and MN animals and their progeny to obtain WT, MN, PN, and DN pups. At 17.5–18.5 dpc, all possible genotypes, including DN, and both genders were present with no significant difference from the expected Mendelian distributions. Survival to adulthood was significantly reduced among MN and DN animals (P<0.001), with reduction in DN more severe. The effect of PN on survival was unclear. Location, shapes, and domains of myonuclei and alignment, continuity, branching pattern, and number and diameter of the muscle fibers were compared between MN and DN siblings, using paraffin‐embedded, hematoxylin and eosin‐stained sections of extensor digitorum longus (EDL) fast‐twitch and soleus (SOL) slow‐twitch muscles. DN muscles had no detectable deficiencies compared to the muscles of MN (N = 7 sibling sets of each muscle type). mRNA levels were compared across all 4 genotypes using NimbleGen Systems 60‐mer expression arrays, 2 arrays per genotype. Two biologically independent cDNA samples were prepared per genotype, each from pools of RNA isolated from hindlimb muscle of 5–6 mice. cDNA labeling, array hybridization, and initial normalization of data were carried out by Nimblegen. Subsequent analysis using ArrayStar software required substantial accommodation for scatter. 4417 calls (~2873 genes) with >2‐fold difference in expression level between the two arrays of any one genotype were removed from analysis and the remaining 38,169 calls (~24,831 genes) were rescaled by quantile ranking. Of genes eliminated, ~135 were involved in muscle function (Panther Database, PD), but only 2 of the 135 were eliminated because of scatter unique to the DN arrays. Thus increased scatter of muscle gene expression cannot be attributed specifically to the DN genotype. Four genes used for normalization of mRNA studies in muscle, Hprt1, Gapdh, Sf3a1, and Hadha, showed consistent levels of expression across all 4 genotypes. mRNAs from the different members of the myosin heavy chain gene family showed the expected diversity in levels of expression. Only 7 genes passed our multi‐step screening for differential expression with a >2.2 fold difference in expression between any 2 genotypes, including VIP neuropeptide hormone, and Myosin heavy chain 4 (Myh4), both involved in muscle contraction. Myh4 mRNA was elevated in MN and DN mice. Vip mRNA was elevated in the DN relative to PN but less relative to MN and WT. Thus, we found no convincing support for a model in which p53 and MyoD act as back‐ups for each other during prenatal myogenesis. It would be of interest to determine the effect of the DN on muscle regeneration and neuromuscular development, which have been shown to be abnormal in the MN mouse. Animal studies were performed in accordance with Cal State LA IACUC 06‐5.Support or Funding InformationCal State LA MBRS‐RISE 1 R25 GM61331 to C. Gutierrez; NIH‐AREA Grant #R15 AR51322‐01 to Sandra B. Sharp