Retention of Pax3 expression in satellite cells of muscle spindles

Abstract

Intrafusal fibers within muscle spindles retain features characteristic of immaturity, unlike the larger and more numerous extrafusal fibers constituting the bulk of skeletal muscle. Satellite cells (SCs), myogenic progenitors, are detected on the surfaces of both intrafusal and extrafusal fibers, but little is known of spindle SCs. We have recently demonstrated that, like their extrafusal counterparts, SCs in muscle spindles of posthatch chickens express paired box transcription factor 7 (Pax7) protein. During vertebrate embryogenesis, myogenic progenitors express both Pax7 and Pax3 proteins. In postnatal mice, Pax3 appears in rare SC subsets, whereas Pax7 is expressed by all SCs within extrafusal fibers. Here we test the hypothesis that Pax3 protein maintains localized expression within SCs of muscle spindles. Immunohistochemical techniques were used to identify SCs by their Pax7 expression within anterior latissimus dorsi muscle excised from posthatch chickens of various ages. A greater percentage of SCs express Pax3 within intrafusal than extrafusal fibers at each age, and the proportion of SCs expressing Pax3 declines with aging. This is the first study to localize Pax3 expression in posthatch avian muscle and within SCs of muscle spindles. We suggest that Pax3-positive SCs are involved in fiber maintenance.

Figure 1

Immunohistochemical labeling of serial cross-sections of anterior latissimus dorsi muscle: 9 days (A–D), and 30 days (E–H) posthatch. In each series, the first image (A or E) is from a serial section adjacent to the cross-section from which the subsequent three images (B–D or F–H) are taken. In the first image (A or E), laminin is red and myosin green. In subsequent images, laminin is deep blue and nuclei are light blue (B–D or F–H), Pax7 is red (C or G), and Pax3 is green (D or H). Several intrafusal fibers clustered within a spindle capsule are near the center of each image, and are smaller in diameter than the larger extrafusal fibers that are always situated outside of the capsule. Open/large arrows indicate intrafusal fibers; small arrows show satellite cells; small arrows with line show a satellite cell expressing Pax3; asterisk, capsule of muscle spindle; e, extrafusal fiber. The same representative intrafusal fibers, extrafusal fibers, and satellite cell nuclei are labeled in each image throughout each series (A–D, E–H). Bar = 30 μm.

Figure 2

Immunohistochemical labeling of serial cross-sections of anterior latissimus dorsi muscle: 62 days (A–D), and 145 days (E–H) posthatch. In each series, the first image (A or E) is from a serial section adjacent to the cross-section from which the subsequent three images (B–D or F–H) are taken. In the first image (A or E), laminin is red, and myosin is green. In subsequent images, laminin is deep blue, and nuclei are light blue (B–D or F–H), Pax7 is red (C or G), and Pax3 is green (D or H). Labeling scheme is the same as that used in Figure 1. Bar = 30 μm.

Figure 3

Pax7-positive/Pax3-positive and Pax7-positive/Pax3-negative cells detected by immunohistochemical labeling of serial cross-sections of anterior latissimus dorsi muscle from a 62-day posthatch chicken. (A) Laminin is deep blue, nuclei are light blue, and Pax7-labeled nuclei are red. (B) Laminin is deep blue, nuclei are light blue, and Pax3-labeled nuclei are green. Curved arrow indicates a myonucleus expressing Pax3. The rest of the labeling scheme is the same as that used in Figure 1. Bar = 20 μm.

Figure 4

Anterior latissimus dorsi muscle weight and fiber size data. Muscle weight (A) increases with age. Ellipse minor axis (B) of extrafusal fibers is greater than that of intrafusal fibers. Ellipse minor axis of extrafusal fibers increases with age but remains constant in intrafusal fibers. Within each graph (A or B), symbols with the same lowercase letter are not significantly different (p>0.05) from one another. Symbols with different lowercase letters are significantly different (p≤0.05) from one another. Each value is expressed as mean ± SE (n=5). Error bars that are not visible are smaller than the symbol used to denote each mean.

Figure 5

Anterior latissimus dorsi muscle intrafusal and extrafusal fiber nuclei data. Satellite cell (SC) frequency (A) is greater in intrafusal than extrafusal fibers at each age studied and, with aging, decreases in both intrafusal and extrafusal fibers. The percentage of SCs expressing Pax3 (B) is also higher in intrafusal than extrafusal fibers at each age studied and also decreases with age. The percentage of myonuclei (MN) expressing Pax3 (C) is initially higher in intrafusal than extrafusal fibers but decreases with age to be statistically the same in intrafusal and extrafusal fibers. Within each graph (A–C), symbols with the same lowercase letter are not significantly different (p>0.05) from one another. Symbols with different lowercase letters are significantly different (p≤0.05) from one another. Symbols with more than one lower case letter are not significantly different from other symbols that bear one or more of the same letters. Each value is expressed as mean ± SE (n=5). Error bars that are not visible are smaller than the symbol used to denote each mean.