Figure 1.
Organization of the DLK1-DIO3 gene cluster in mammals.
The relative positions of the maternal non-coding RNA genes and paternal protein coding genes are based on the bovine chromosome 21 sequence contig NW_001494068. The genes are shaded black on the parent-of-origin chromosome to indicate their allele-specific expression. The protein coding genes are expressed from the paternal allele (pat) and the non-coding RNA are expressed from the maternal allele (mat). The causative mutation (SNPCLPG) for the callipyge phenotype lies in the intergenic region between DLK1 and MEG3. The black diamond indicates the approximate position of Affymetrix probe Bt18078.1.S1_at. The presence of C/D snoRNA and most miRNA have been detected in the bovine genome sequence but have not been confirmed in sheep.
Figure 2.
Muscle weight changes in callipyge (+/C) and normal (+/+) lambs.
Muscle weights were regressed on live weight for the longissimus dorsi (A), semimembranosus (B) and supraspinatus (C). Equations are given for each muscle and genotype with an asterisk indicating statistical significance (P<.0001) in slopes and overall equations. There were no effects of genotype on the intercepts of any regression lines. The longissimus dorsi and semimembranosus muscles of callipyge lambs grew at a faster rate that normal lambs, but growth of the supraspinatus was not affected by genotype.
Figure 3.
Myofiber area of semimembranosus muscles in callipyge and normal lambs.
Cross sectional areas of fast and slow twitch myofibers of 20–30 day old callipyge and normal lambs are shown. Differing lower case letters indicate significance between genotypes (P = 0.0009). There was a trend (P = 0.0804) for the area of slow twitch fibers to be larger in callipyge lambs. These results indicated hypertrophic growth had begun in callipyge lambs at these ages.
Table 1.
Summary of functional annotation clusters for the effect of genotype.
Figure 4.
Hierarchal clustering and microarray signal intensity for genes validating an effect of genotype by quantitative PCR.
Gene names are listed for rows and columns are fixed for age in days and genotype (+/C for callipyge and +/+ for normal). Relationship amongst genes sets was determined by hierarchal clustering in the heatmap.2 function of Bioconductor. Relative intensity by RMA is represented by green if expression was higher than average or red if expression was lower than average.
Table 2.
Affymetrix probe sets that were validated by quantitative PCR.
Figure 5.
Candiate genes with an expression pattern resembling DLK1.
Least square means and standard errors for log transcript abundance in 100 ng of total RNA in a hypertrophied muscle (semimembranosus, A, C, E, G) and a non-hypertrophied muscle (supraspinatus, B, D, F, H) in callipyge (+/C) and wild-type (+/+) lambs. Birth is day 0 and samples from 14 days prepartum are included and represented as -14 days. DLK1 expression is shown in A and B. Transcripts PDE4D (C and D), PARK7 (E and F) and BHLHB3 (G and H) show a significant effect of the callipyge genotype in semimembranosus, while no effect is observed in the supraspinatus. This pattern of expression suggests these genes are responding to DLK1 signaling in callipyge muscle.
Figure 6.
PDE7A exhibits an expression pattern resembling RTL1.
Least square means and standard errors for log transcript abundance in 100 ng of total RNA in a hypertrophied muscle (semimembranosus, A and C) and an unaffected muscle (supraspinatus, B and D) in callipyge (+/C) and wild-type (+/+) lambs. Birth is day 0 and samples from 14 days prepartum are included and represented as -14 days. RTL1 expression is shown in A and B and PDE7A expression is shown in C and D. Transcript abundance of RTL1 and PDE7A is up-regulated as a result of the callipyge allele in both muscles, suggesting PDE7A may respond to RTL1 expression.
Figure 7.
Expression of MEG8 and CB439344 in the semimembranosus influenced by the presence of a maternal callipyge allele.
Expression of MEG8 exons in all four possible callipyge genotypes (A) is shown in comparison to expression of transcript CB439344 (B) that maps to the C/D snoRNA clusters of MEG8 in cattle. Lambs possessing a maternal callipyge allele (C/+ and C/C) exhibit significantly higher levels of MEG8 expression and CB439344 than callipyge (+/C) and normal lambs (+/+).
Figure 8.
Influence of PARK7 and TXNIP on AKT/mTOR signaling in callipyge muscle hypertrophy.
The diagram depicts the AKT response to IGF-1 signaling which increases protein synthesis. Two regulatory proteins of the AKT/mTOR pathway are differentially expressed in callipyge hypertrophied muscles. Factors that are inhibitory of protein synthesis are indicated in red and stimulatory factors are green. Transcripts that were down-regulated in callipyge (+/CPat) muscle are rectangles and transcripts that were up-regulated in callipyge muscle are cross shaped. In this model, the phosphatase activity of PTEN is inhibited by PARK7 and TXNIP and the AKT/mTOR response to normal physiological levels of growth factors is magnified to increase protein synthesis rates.