Pax7 −/− mice develop megaesophagus

Most Pax7 −/− mice die within 3 weeks of birth [20]. Surviving P21 Pax7 −/− mice displayed megaesophagus with 100 % penetrance. P21 Pax7 −/− esophagi had an enlarged diameter relative to Pax7 +/+ and Pax7 +/− esophagi, and a dilated lumen filled with partially digested food (Fig. 1a–d). No other gross abnormalities were observed in the GI tracts of Pax7 −/− mice. P21 Pax7 +/− esophagi resembled the wild type in esophageal length, location of the skeletal-smooth muscle boundary, and luminal diameter (Additional file 1: Figure S1); therefore, Pax7 +/+ and Pax7 +/− esophagi were used interchangeably as controls. Immunofluorescence analysis (IFA) of longitudinal sections of P21 esophagi with markers of smooth muscle (α-smooth muscle actin (αSMA)) and skeletal muscle (sarcomeric actin (SA)) revealed that Pax7 −/− mice have a mispatterned ME, with the skeletal-smooth muscle boundary occurring at an abnormally proximal position (Fig. 1c, d). Most smooth muscle in the esophagi of Pax7 +/− mice was found in a short, broad segment at the esophagogastric junction, where SMCs were bundled into fascicles that were stacked side by side with an orientation perpendicular to the lumen (Fig. 1i). In contrast, distal to the aberrantly proximal boundary in Pax7 −/− esophagi, there was a long, thin, ectopic extension of smooth muscle that connected to the LES (Fig. 1d, j). The fascicles in this ectopic region of smooth muscle in Pax7 −/− esophagi were parallel to the lumen, indicating they had failed to undergo the normal reorientation process. A few SMCs were dispersed within the skeletal muscle at the skeletal-smooth muscle boundary in both Pax7 +/− and Pax7 −/− esophagi (Fig. 1g, h). Despite the mispatterned and thinner ME distal to the aberrantly proximal boundary in Pax7 −/− esophagi, the morphology of the smooth muscle fascicles appeared normal in the Pax7 −/− LES (Fig. 1k, l). The Pax7 −/− LES was somewhat thinner than that of the control mice (control = 135.8 ± 9.6 mm (n = 5); mutant = 92.0 ± 12.9 mm (n = 6); p < 0.01). This may be due to a combination of defective development of the ME and the overall growth retardation displayed by these mice [20]. The ME of the proximal portion of Pax7 −/− esophagi was similar to that of Pax7 +/− esophagi, although the skeletal myofibers in mutants appeared to have a greater amount of between-fiber space than in controls (Fig. 1f).

Fig. 1 Pax7 −/− mice have megaesophagus. a, b P21 Pax7 +/− and Pax7 −/− esophagi. c–j Longitudinal sections of P21 Pax7 +/− and Pax7 −/− esophagi were stained with antibodies to α-smooth muscle actin (αSMA; red) and sarcomeric actin (SA; green) for smooth and skeletal muscles, respectively. c, d Pax7 −/− esophagi have an aberrantly proximally located skeletal-smooth muscle boundary (arrows). Note that the Pax7 +/+ esophagus is cut off at the top end, making it appear shorter than the Pax7 −/− esophagus, the full length of which is shown. In fact, at P21, Pax7 −/− esophagi are shorter than Pax7 +/+ esophagi (see Fig. 3e). e, f The proximal ME of Pax7 −/− esophagi has skeletal muscle of relatively normal appearance. g, h Smooth muscle cells are dispersed with skeletal myofibers around the skeletal-smooth muscle boundary (Boundary). Note that the ME of the Pax7 −/− esophagus is thinner than the control at the skeletal-smooth muscle boundary. i, j The distal Pax7 −/− esophagus has a long, thin extension of smooth muscle instead of the short, broad segment of smooth muscle found in controls. e–j The thin αSMA+ layer adjacent to the ME is the muscularis mucosa (arrowheads). k, l Longitudinal sections through the LES of P21 Pax7 +/+ and Pax7 −/− esophagi mice were stained with antibodies to αSMA (green) and nNOS (red) to label inhibitory intramural neurons. m Quantification of the relative numbers of nNOS+ cells to αSMA staining in the ME of the LES. Values are means ± SD, n = 5. Bars: (c, d) 1 mm; (e–l) 0.2 mm Full size image

Megaesophagus is sometimes associated with loss of nitrergic neurons in the myenteric plexus of the LES [3]. We therefore quantified the number of neurons in the LES from P21 Pax7 +/+ and Pax7 −/− mice. IFA of LES sections with an antibody to nNOS revealed that the number and pattern of nitrergic neurons were similar between control and mutant mice (Fig. 1k–m). Taken together, P21 Pax7 −/− mice showed megaesophagus with an aberrantly proximal skeletal-smooth muscle boundary. The morphological defects of the P21 Pax7 −/− ME were most apparent in the region between the skeletal-smooth muscle boundary and LES, but were not as severe in the proximal region and in the LES itself.

Defective ME patterning in Pax7 −/− esophagi

To determine when these defects in Pax7 −/− mice arose, cross sections were taken from P0-P21 at proximal, mid-level, and distal regions. Expression of αSMA and SA was assessed. The proximal and distal ME were composed of skeletal and smooth muscles, respectively, and the cross sections of proximal and distal Pax7 −/− esophagi showed no obvious defects at P0, P7, and P21 (Fig. 2a–f, m–r, s, u). Mid-level cross sections revealed that esophageal dilation became recognizable in Pax7 −/− esophagi at P7 (Fig. 2g–j, t), and this distention was more severe at P21 (Fig. 2k, l, t). H&E-stained cross sections revealed that the P21 Pax7−/− ME was thinner than that of controls in the mid-level region (Fig. 2v–x). In contrast to the control mid-level ME, which was composed of skeletal muscle at P7 and P21, the Pax7 −/− mid-level ME displayed only smooth muscle at these time points (Fig. 2i–l).

Fig. 2 Defects in ME patterning in Pax7 −/− esophagi. a–r Cross sections of P0, P7, and P21 Pax7 +/− and Pax7 −/− esophagi were stained with antibodies to αSMA, SA, and DAPI. j, l The P7 and P21 Pax7 −/− esophagi are dilated, and skeletal muscle is replaced by smooth muscle at the mid-level region (Mid). s–u Luminal diameter at the mid region of Pax7 −/− esophagi is larger than that of Pax7 +/− esophagi at P7 and P21. Values are means ± SD, n = 3–11. v, w Cross sections of P7 Pax7 +/− and Pax7 −/− esophagi at the mid region were stained with H&E. Ep epithelium, MM muscularis mucosa, ME muscularis externa. The black horizontal bars indicate the width of the ME; the asterisks indicate the lumens. x Quantification of the width of the ME, as shown in (v and w). Values are means ± SD, n = 3. Bars: (a–r) 0.2 mm, (v, w) 50 μm. **P < 0.01; ***P < 0.001 Full size image

These findings are consistent with the aberrant skeletal-smooth muscle boundary seen in P21 Pax7 −/− esophagi (Fig. 1d), and they suggested a defect in the process whereby smooth muscle is replaced by skeletal muscle in these animals. We therefore analyzed the proximal-to-distal progression of skeletal myogenesis during ME development. Expression of αSMA and SA was assessed in longitudinal sections taken at P0, P7, and P21 (Figs. 1c, d and 3a–d). The distal-most skeletal muscle cells in these sections were embedded in smooth muscle. Progression of ME development was monitored by measuring the distance from the distal-most SA+ cell to the LES. At P0, this distance was about half of the length in Pax7 +/− esophagi, whereas it constituted nearly 80 % of the total length in Pax7 −/− esophagi (Fig. 3a, b, e). The distance from the distal-most SA+ cell to the LES diminished in Pax7 +/− esophagi from P0 to P21, and the distal-most SA+ cell was found just proximal to the LES at P21 (Figs. 1c and 3a, c, e). In contrast, the distal-most SA+ cell in Pax7 −/− animals was at a significantly more proximal location than in Pax7 +/− animals as early as P0 and maintained through P21 (Figs. 1d and 3b, d, e). The distance from the distal-most SA+ cell to the LES slightly decreased in Pax7 −/− esophagi by P21; however, the distance still constituted ~65 % of the total length (Fig. 3e). The total length of Pax7 −/− esophagi is ~20 % shorter than that of control animals by P21 (Fig. 3e) [14]. This is likely due to overall growth retardation in these mice [20], but cannot alone account for the ME patterning defects.

Fig. 3 Pax7 −/− mice display defective proximal-to-distal progression of ME development. a–d Longitudinal sections of P0 and P7 Pax7 +/− and Pax7 −/− esophagi were stained with antibodies to αSMA, SA, and DAPI. Pax7 −/− esophagi have an aberrantly proximally located skeletal-smooth muscle boundary (arrows). The SA+ tissue near the distal end of the Pax7 +/− esophagus in (a) is the diaphragm. e The distance between the distal-most SA+ cell and the LES was measured and is represented by the red portion of the histogram bars. The distance decreases progressively with age in Pax7 +/− esophagi, but this fails to occur in Pax7 −/− esophagi. Values are means ± SD, n = 3–5. Bars: 1 mm. ***P < 0.001 Full size image

Pax7 −/− esophagi have reduced number of cells undergoing myogenic differentiation

To analyze the Pax7 expression pattern in developing esophageal ME, longitudinal sections of Pax7 +/− esophagi were taken at P7 and P21 and stained with an antibody for Pax7. At P7, most Pax7+ cells were found in the TZ, and their number decreased at more proximal locations (Fig. 4a, c, e, g). Pax7+ cells diminished from the entire esophagi at P21, when proximal-to-distal progression of skeletal muscle was complete (Fig. 4b, d, f, g). At this stage, relatively few Pax7+ cells were present, as these probably mainly represent the population of satellite cells seen in the adult esophagus [13, 24].

Fig. 4 Expression of Pax7 in the esophageal musculature at P7 and P21. a–f Longitudinal sections of P7 and P21 Pax7 +/− esophagi were stained with antibodies to Pax7, laminin, and DAPI. Proximal, mid-level (Mid), and TZ regions were analyzed. The greatest number of Pax7+ cells was observed in the TZ at P7, and the numbers diminished in mid and proximal regions. The number of Pax7+ cells is diminished at P21 when proximal-to-distal progression of ME development is complete. g Quantification of Pax7+ cells in proximal (prox), mid, and TZ regions measured as a percentage of total DAPI+ cells in the ME. Bars: 0.2 mm Full size image

We hypothesized that impaired proximal-to-distal progression of skeletal myogenesis in Pax7 −/− esophagi resulted from a decrease in the number of cells undergoing myogenic differentiation. We therefore assessed the expression of MyoD (a marker of determined myoblasts) and myogenin (a marker of differentiating myoblasts) during ME development. Longitudinal sections of P7 Pax7 +/− and Pax7 −/− esophagi were stained with antibodies for MyoD and myogenin. Consistent with our previous study [13], most MyoD+ and myogenin+ cells were found in the TZ and their numbers diminished at more proximal locations in control animals. Pax7 −/− mice had ~30 % the number of MyoD+ cells and ~50 % the number of myogenin+ cells as control mice (Fig. 5a–f).

Fig. 5 Reduced skeletal muscle precursor cell proliferation in the Pax7 −/− TZ. a, b, d, e Longitudinal sections of P7 Pax7 +/− and Pax7 −/− esophagi were stained with either MyoD (a, b) or Myogenin (d, e) antibodies and DAPI. The TZ was analyzed. c, f Quantification of MyoD+ (c) and Myogenin+ (d) cells within the TZ, measured as a percentage of total DAPI+ cells in the ME. Values are means ± SD, n = 3–5. g–j, l–o Longitudinal sections of P7 Pax7 +/− and Pax7 −/− esophagi were stained with either Ki67 (g–j) or phospho-histone H3 (PH3) (l–o) and DAPI. The TZ and distal region were analyzed. k, p Quantification of Ki67+ (k) and PH3+ (p) cells within the TZ and distal region, measured as a percentage of total DAPI+ cells in the given region. The numbers of Ki67+ and PH3+ cells were reduced in the TZ in Pax7 −/− mice, but not in the distal region. Values are means ± SD, n = 4–5. q–v Longitudinal sections of P7 Pax7 +/− and Pax7 −/− esophagi were stained with MyoD and Ki67 antibodies and with DAPI. w Quantification of MyoD+/Ki67+ cells within the TZ, measured as a percentage of total DAPI+ cells in the TZ. Values are means ± SD, n = 4. x–cc Longitudinal sections of P7 Pax7 +/− and Pax7 −/− esophagi were stained with M-cadherin (Mcad) and Ki67 antibodies and with DAPI. dd Quantification of total Mcad+ and Mcad+/Ki67+ cells within the TZ, measured as a percentage of total DAPI+ cells in the TZ. Values are means ± SD, n = 4. Bars: 0.2 mm (a–v); 20 μm (x–cc). *P < 0.05; **P < 0.01 Full size image

Reduction of the number of cells expressing these markers of myogenesis may result from a decrease in the muscle progenitor cell population, either by alteration in cell proliferation or cell survival. To investigate cell proliferation in the developing ME, longitudinal sections were taken from P7 Pax7 +/− and Pax7 −/− esophagi and stained with antibodies for Ki67 and phospho-histone H3. Most Ki67+ and phospho-histone H3+ cells were found in the TZ in Pax7 +/− esophagi (Fig. 5g, k, l, p). Much lower numbers of Ki67+ and phospho-histone H3+ cells were found in the Pax7 −/− TZ (Fig. 5h, k, m, p). Consistent with the largely postmitotic nature of ME smooth muscle cells [13], fewer Ki67+ and phospho-histone H3+ cells were present in the distal ME below the TZ in both Pax7 +/− and Pax7 −/− esophagi (Fig. 5i, j, k, n, o, p). Therefore, cell proliferation in the TZ is substantially reduced in Pax7 −/− mice.

To confirm that cell proliferation was reduced in skeletal muscle precursor cells per se, we stained longitudinal P7 sections with antibodies for MyoD or another lineage marker, M-cadherin, plus Ki67. Pax7 −/− mice had ~20 % the number of MyoD+/Ki67+ cells in the TZ as control mice (Fig. 5q–w). Pax7 −/− mice had only ~13 % the number of M-cadherin+ cells as controls (Fig. 5x–dd). Furthermore, ~45 % of control M-cadherin+ cells were also positive for Ki67, whereas none of the small number of M-cadherin+ cells in the Pax7 −/− TZ were also positive for Ki67. These results indicate that cell proliferation of skeletal muscle precursors is strongly diminished in the Pax7 −/− esophagus.

We next analyzed longitudinal sections of P7 Pax7 +/− and Pax7 −/− esophagi by TUNEL assay and by IFA with an antibody to cleaved caspase-3, with thymuses as positive controls. TUNEL+ and cleaved caspase-3+ cells were easily detected in control thymuses (Additional file 2: Figure S2E, J). Consistent with previous studies [7–9, 13], no apoptotic cells were observed in Pax7 +/− esophagi (Additional file 2: Figure S2A, C, F, H). Furthermore, no TUNEL+ or cleaved caspase-3+ cells were observed in the esophagi of Pax7 −/− mice (Additional file 2: Figure S2B, D, G, I), indicating that the diminished skeletal myogenesis seen in these mice is not a consequence of cell death.