PAX7 is required for patterning the esophageal musculature
© Chihara et al. 2015
Received: 23 June 2015
Accepted: 7 November 2015
Published: 3 December 2015
The mammalian esophageal musculature is unique in that it makes a transition from smooth to skeletal muscle, with most of this process occurring after birth. In order to better understand the mechanisms that control esophageal musculature development, we investigated the roles in this process of the paired box transcription factor, PAX7, a principal regulator of skeletal myogenic progenitor cells. Previous studies showed that Pax7 is important for determining the esophageal muscle composition.
We characterized the postnatal development of the esophageal musculature in Pax7 −/− mice by analyzing morphology, muscle composition, and the expression of markers of myogenesis, cell proliferation, and apoptosis. Pax7 −/− mice displayed megaesophagus with a severe defect in the postnatal developmental process whereby esophageal smooth muscle is replaced by skeletal muscle. Pax7 −/− esophagi have substantially reduced skeletal muscle, most likely due to diminished proliferation and premature differentiation of skeletal muscle precursor cells. This impaired the proximal-to-distal progression of skeletal myogenesis and indirectly affected the patterning of the smooth muscle-containing portion of the esophageal musculature.
Postnatal patterning of the esophageal musculature appears to require robust, PAX7-dependent cell proliferation to drive the proximal-to-distal progression of skeletal myogenesis. This process in turn influences distal smooth muscle morphogenesis and development of the mature pattern of the esophageal musculature.
KeywordsEsophageal myogenesis Megaesophagus Pax7 Skeletal muscle Smooth muscle Cell proliferation Frontal expansion
The musculature of the esophagus controls passage of food into the stomach by waves of peristaltic contractions. The lower esophageal sphincter (LES) is a bundle of smooth muscles at the distal end of the esophagus, where it meets the stomach. During swallowing, tonic smooth muscles of the LES relax briefly in order to allow passage of food into the stomach . Esophageal muscles are adversely affected in a number of human disorders, including myotonic dystrophy, oculopharyngeal muscular dystrophy, and the inflammatory myopathies; this may lead to dysphagia, regurgitation, choking while eating, and other symptoms. Disorders of LES function include gastroesophageal reflux disease (GERD) and achalasia, the latter characterized by impaired relaxation of the LES and perturbed peristalsis, often resulting in megaesophagus. The primary cause of achalasia is defective signaling between the NO-producing inhibitory myenteric neurons and neighboring smooth muscle cells (SMCs) of the LES [2, 3]. Although studies have identified several mutant mouse lines that display these esophageal defects, their etiologies are poorly understood.
The mammalian esophageal musculature is unique in that it makes a transition from smooth to skeletal muscle, with most of this process occurring after birth. The esophagus is ensheathed by the muscularis externa (ME) [1, 4, 5]. Skeletal muscle comprises the proximal portion of the ME and is critical for swallowing and proximal waves of peristalsis. Smooth muscle surrounds the distal portion, including the esophagogastric junction, which harbors the LES. During mammalian development, the ME initially comprises only smooth muscle. In the mouse, skeletal muscle precursors are first detected in the proximal ME at embryonic day (E) 13 . Over the next 3 weeks of life, smooth muscle is replaced by skeletal muscle in a proximal-to-distal manner, and the adult ME pattern is nearly completed by postnatal day (P) 14 [7–9]. Human esophageal myogenesis is similar, although smooth muscle is maintained in a more proximal position than in the mouse, but is still restricted to the distal one-third of the esophagus . Interestingly, the amount and distribution of skeletal muscle varies among individuals [11, 12]. The developmental and cell biological mechanisms that underlie the smooth to skeletal muscle replacement process have been controversial, with multiple mechanisms proposed [7–9, 13–15] (reviewed in ref. ). Rishniw et al.  were the first to suggest that some sort of distal compaction of smooth muscle cells was important in the replacement of smooth muscle with skeletal muscle. However, the mechanisms for this process remained unclear.
Our previous study provided insight into mechanisms that control morphogenesis of the esophageal ME . We showed that mice lacking the multifunctional cell surface receptor Cdo (also called Cdon) have a defect in the postnatal developmental process whereby esophageal smooth muscle is replaced by skeletal muscle. Analyses of various skeletal myogenic markers revealed that proliferative skeletal muscle progenitor cells migrated in a transition zone (TZ) along the length of the esophagus in a proximal-to-distal manner, leaving differentiated myofibers in its wake. Distal to the TZ, smooth muscle fascicles underwent a morphogenetic process whereby they changed their orientation relative to each other and to the lumen. In a distal-to-proximal manner, smooth muscle fascicles altered their orientation from parallel to the lumen to nearly perpendicular to the lumen. Upon completion of the process, smooth muscle occupied only the most distal part of the esophagus and the LES. Cdo −/− mice had no defect in skeletal myogenesis or in the numbers of various skeletal muscle progenitor cell types in the TZ. Rather, they were specifically defective in SMC fascicular reorientation, leading to ectopic, proximal fascicles displaying an inappropriate orientation and an aberrantly proximal skeletal-smooth muscle boundary . Importantly, Cdo −/− mice also displayed megaesophagus and achalasia, with a smooth muscle cell-intrinsic defect in NO-mediated LES relaxation. Taken together, our previous findings identified a mechanism of ME maturation and provided a link between esophageal morphogenesis and motility disorders. Although the ME maturation defect in Cdo −/− mice is almost certainly smooth muscle cell-autonomous, a role for TZ-based skeletal myogenesis in promoting the fascicular reorientation process seems likely.
Multiple mechanisms have also been proposed for the developmental origin of esophageal skeletal muscles (ESMs). Minchin et al. reported that, similar to trunk and limb muscles, ESMs arise from Pax3/Pax7-expressing cells that originate in somites . However, Gopalakrishnan et al. have recently demonstrated that ESMs are derived from the cardiopharyngeal mesoderm, and their development is dependent on expression of Tbx1 and Isl1, genes important for specific muscles of the head but not the trunk . These factors lie upstream of the myogenic bHLH factors, MyoD and myogenin, which promote commitment and differentiation of cells in the skeletal muscle lineage . Consistent with a cranial mesoderm origin for ESMs, Pax3 was not expressed during development of these muscles and was dispensable for their development in mice .
In contrast to Pax3, Pax7 is expressed in skeletal muscle precursors of the esophageal TZ [13, 19–22]. Importantly, however, Pax7 is never expressed in the SMC lineage as shown by lineage tracing studies, immunochemistry, and in situ hybridization [13, 19–22]. Worl et al. used electron microscopy to show that Pax7 −/− mice had a reduced number of skeletal muscle precursor cells in the esophageal ME, defective esophageal skeletal myogenesis, and an aberrantly proximal skeletal-smooth muscle boundary . As Pax7 is not expressed in the smooth muscle lineage, such mice offer an opportunity to ask whether TZ-based skeletal myogenesis might affect smooth muscle patterning in the esophagus. We report here that Pax7 −/− mice have a severe defect in the postnatal developmental process whereby esophageal smooth muscle is replaced by skeletal muscle. Our results suggest that this defect is due to reduced cell proliferation in the TZ, probably due to precocious differentiation of skeletal muscle precursor cells, leading to a deficit in skeletal muscle. This defect in Pax7 −/− mice is associated with both megaesophagus and defective maturation of esophageal smooth muscle patterning. Our findings add to the previous model for esophageal musculature development. PAX7-dependent cell proliferation in the TZ is important for the proximal-to-distal progression of esophageal skeletal myogenesis. Furthermore, loss of PAX7 leads to a non-autonomous defect that influences smooth muscle fascicle reorientation and development of a mature ME.
Pax7 +/− mice were originally obtained from Peter Gruss  and are from the colony maintained by the Rudnicki laboratory. These mice are on a 129Sv background. It has previously been reported that on a C57BL/6J background, Pax7 +/− esophagi have a mild defect in development of the esophageal musculature . On the 129Sv background, we have not observed differences in esophageal development between Pax7 +/+ and Pax7 +/− mice, and at P21 they were indistinguishable in esophageal length, location of the skeletal-smooth muscle boundary, and luminal diameter (Additional file 1: Figure S1). Therefore, we used both Pax7 +/+ and Pax7 +/− animals as controls. All animal procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committees (IACUC) of the Icahn School of Medicine at Mount Sinai and according to the Canadian Council on Animal Care guidelines and the University of Ottawa Animal Care Committee protocols.
Preparation of frozen sections
Dissected esophagi were prepared for histology by directly freezing in OCT. For longitudinal sections, incisions were made along the entire esophagus in order to clear out the ingesta, and the sheet of dissected esophagus was laid flat prior to freezing. Frozen tissue blocks were sectioned at 10 μm and placed on Superfrost Plus slides (Thermo Fisher Scientific).
Immunofluorescence and histology
Frozen sections (10 μm) were immunostained as previously described . Slides were fixed in 4 % PFA, washed in PBS, permeabilized in 0.3 % Triton/PBS, washed in PBS, blocked in 10 % goat serum, and incubated overnight at 4 °C with primary antibodies in blocking buffer. Additional M.O.M. blocking (Vector Laboratories) steps were performed according to the manufacturer’s instructions when mouse primary antibodies were used. Antibodies used included mouse anti-sarcomeric actin (5C5; 1:1000; Sigma-Aldrich), rabbit anti–α-smooth muscle actin (1:200; Abcam), rabbit anti-MyoD (1:500; Santa Cruz Biotechnology, Inc.), mouse anti-myogenin (F5D; 1:50; Santa Cruz Biotechnology, Inc.), mouse anti-M-cadherin (1:75; Santa Cruz Biotechnology, Inc.), rabbit anti-nNOS (C12H1; 1:500; Cell Signaling Technology), rabbit anti-Ki67 (1:1000; Leica), mouse anti-Ki67 (1:100; BD Pharmingen), and rabbit anti-phospho-Histone H3 (ser10) (1:1000; Millipore). After permeabilization, the following antibodies required an antigen retrieval step of boiling the sections in 10 mM sodium citrate: mouse anti-Pax7 (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA), and rabbit cleaved caspase-3 (Asp175; 1:1000; Cell Signaling Technology). Fluorescent secondary antibodies were from Invitrogen and used at 1:1000: goat anti-rabbit Alexa Fluor 488, anti-rabbit Alexa Fluor 568, goat anti-mouse Alexa Fluor 488, and goat anti-mouse IgG1 Alexa Fluor 647. Nuclei were fluorescently labeled with DAPI and mounted with Fluoromount-G anti-fade medium (SouthernBiotech). TUNEL assay was performed using the In Situ Cell Death Detection kit from Roche. For Figs. 4 and 5 and Additional file 2: S2, the transition zone was identified by adjacent longitudinal sections stained with antibodies to 〈-smooth muscle actin and sarcomeric actin.
Hematoxylin and eosin (H&E) staining was performed as described . Sections were dehydrated through graded ethanol and xylene and mounted with Permount (Fisher Scientific).
Microscopy was performed at the Mount Sinai Microscopy Shared Resource Facility using Axioplan2 and Axioplan2 IE microscopes (Carl Zeiss) equipped with ×10, ×20, ×40, and ×100 objective lenses that had numerical apertures of 0.5, 0.8, 0.75, and 1.4, respectively. Images were acquired at room temperature with a camera (Axio Cam MRm; Carl Zeiss). Mosaic images were compiled using the stitching feature in the AxioVision software. The maximum luminal diameter and length of esophageal skeletal/smooth muscle were measured by using ImageJ software (National Institutes of Health).
Data were expressed as mean ± standard deviation. Differences were tested by two-tailed t test. Values with P < 0.05 were considered statistically significant.
Pax7 −/− mice develop megaesophagus
Megaesophagus is sometimes associated with loss of nitrergic neurons in the myenteric plexus of the LES . 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
Pax7 −/− esophagi have reduced number of cells undergoing myogenic differentiation
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 , 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.
In this study, we investigated the role of Pax7 in esophageal musculature development. Pax7 −/− mice displayed megaesophagus with a severe defect in the postnatal developmental processes whereby esophageal smooth muscle is replaced by skeletal muscle and the ME acquires a mature pattern. The proximal-to-distal progression of skeletal myogenesis was impaired in Pax7 −/− esophagi, associated with a significant reduction in cell proliferation of skeletal muscle precursors in the TZ. Additionally, our findings revealed that Pax7 is important for proper postnatal patterning of the entire ME, including the distal smooth muscle layers.
We previously proposed a model for postnatal development of the esophageal ME . During esophageal ME maturation, skeletal muscle progenitors commit to the myogenic lineage and differentiate within a TZ that migrates along the length of the esophagus in a proximal-to-distal manner. Distal to the TZ, SMCs are largely non-proliferative and bundled into long, thin fascicles. SMC fascicles are initially arranged end-to-end and parallel to the lumen. During ME maturation, SMC fascicles rearrange their orientation such that they are ultimately arranged side by side and nearly perpendicular to the lumen; as a consequence, at the end of ME maturation, the fascicles occupy only the distal portion of the ME. Fascicular reorientation occurs in a distal-to-proximal manner. We suggested that this process “clears a path” for the TZ, which migrates distally and produces the skeletal myofibers that constitute the majority of the length of the mature ME of the adult mouse .
Mice lacking the multifunctional cell surface receptor, Cdo, have a mispatterned ME, including an aberrantly proximal skeletal-smooth muscle boundary, and they develop megaesophagus. These mice are specifically defective in SMC fascicular reorientation, without obvious alterations in TZ-based skeletal myogenesis . Cdo −/− mice had an ectopic, proximal region of smooth muscle in which the fascicles remained in the end-to-end, parallel-to-the-lumen orientation that normally characterizes an earlier developmental stage. This phenotype is very likely to be SMC-autonomous . In contrast, the esophageal defects in Pax7 −/− mice are almost certainly autonomous to the skeletal muscle lineage. Pax7 is expressed in skeletal muscle precursor cells throughout the body, including the esophageal TZ, but it is never expressed in the SMC lineage [13, 19–22]. Examination of esophageal ME patterning in Pax7 −/− esophagi revealed that their musculature consisted mainly of smooth muscle, having skeletal muscle only at the very proximal end. This phenotype was more severe in Pax7 −/− esophagi than Cdo −/− esophagi. Smooth muscle occupied the distal ~20 % of P14 Cdo −/− esophagi, whereas it occupied the distal ~65 % of P21 Pax7 −/− esophagi. There was no difference in the position of the skeletal-smooth muscle boundary between control and Cdo −/− mice at P0, but Pax7 −/− esophagi already had an aberrantly proximal skeletal-smooth muscle boundary at P0. Moreover, in contrast to Cdo −/− mice, the numbers of MyoD+ and myogenin+ cells were reduced, as was cell proliferation, in the Pax7 −/− TZ. Therefore, the aberrantly proximal skeletal-smooth muscle boundary in Pax7 −/− esophagi resulted from reduced production of ESM.
Distal to the aberrantly proximal skeletal-smooth muscle boundary, the Pax7 −/− smooth muscle layer was very thin, with the fascicles parallel to the lumen for nearly the entire length of the ME. The LES was normally patterned, consistent with it being in the region in which fascicles reorient earliest . As Pax7 is never expressed in the SMC lineage, the mispatterning of ESM in Pax7 −/− esophagi is presumably a non-cell-autonomous effect. It seems likely that smooth muscle mispatterning occurs as a consequence of impaired proximal-to-distal progression of the Pax7 −/− TZ, but further work will be required to prove this. One physiological consequence of this combination of deficient skeletal myogenesis and SMC fascicle mispatterning is that Pax7 −/− mice have an enlarged luminal diameter specifically in the mid region of the esophagus (i.e., megaesophagus). We speculate that smooth muscles, in this abnormal location and fascicular arrangement, are not successful in maintaining the full integrity and function of the ME. This may occur for a variety of non-mutually exclusive reasons, including defective peristalsis, causing the slow passage of food and physical distension; suboptimal innervation; or some combination of these and additional processes.
Our findings support and add to the previous model for esophageal musculature development. Based on the fact that Pax7 −/− mice display a presumably non-SMC-autonomous defect in fascicular reorientation, we propose the following hypothesis. Smooth muscle fascicular reorientation may be facilitated by forces exerted via the proximal-to-distal progression of the TZ. This progression may “push” the SMCs to a more distal region, where they are eventually exposed to the signals that trigger the rearrangement of SMCs relative to one another, resulting in reorientation of fascicles. Proliferation of skeletal muscle precursor cells was significantly reduced in the Pax7 −/− TZ during esophageal muscular development. This suggests that robust cell proliferation, which presumably participates in driving the proximal-to-distal progression of the TZ, depends on PAX7. This is consistent with the known roles of PAX7 in maintaining proliferation and preventing precocious differentiation [24, 25]. Therefore, it is most likely that skeletal muscle precursor cells differentiate prematurely during esophageal myogenesis in the Pax7−/− TZ, resulting in reduced production of ESM.
That robust cell proliferation in the TZ may be critical for the proximal-to-distal progression of skeletal myogenesis is supported by a mathematical model of cell migration . This model predicts that proliferation at the invading front is the key mechanism driving directed migration. These predictions were experimentally validated with the directional migration and colonization of the gut by vagal neural crest cells that establish the enteric nervous system . This “frontal expansion” model, which emphasizes the importance of proliferation to a carrying capacity, is potentially relevant to a wider range of invasion systems such as angiogenesis, epidermal wound healing, and malignant invasion . Therefore, this model may also apply to the proximal-to-distal progression of esophageal skeletal myogenesis.
Megaesophagus is observed as a feature of several animal models of Duchenne muscular dystrophy, including some strains of mdx mice, and some dystrophic dog and cat models [27–30]. It was proposed that megaesophagus in some of these animal models was caused by a hypertrophic diaphragm and ensuing esophageal constriction, a very different scenario than that seen in Pax7 −/− mice. There is, however, little in the literature to suggest that megaesophagus is common in patients with muscular dystrophy or other myopathies. Megaesophagus occurs more frequently in severe cases of achalasia, as a consequence of inability to fully relax the LES [2, 3]. It will be of interest for future studies to use electrophysiology and manometry to test if Pax7 −/− mice have a non-autonomous defect in LES function.
Our analyses on Pax7 −/− esophageal musculature development suggest that ME patterning requires robust, PAX7-dependent cell proliferation in the TZ, which helps drive the proximal-to-distal progression of skeletal myogenesis. This process may in turn be necessary for distal smooth muscle fascicle reorientation and development of a mature ME.
cell adhesion molecule-related/down-regulated by oncogenes
esophageal skeletal muscle
gastroesophageal reflux disease
lower esophageal sphincter
myogenic factor 5
myoblast determination protein
neuronal nitric oxide synthases
paired box protein
smooth muscle actin
smooth muscle cell
This work was supported by a grant from the NIH (AR46207) to RSK; grants from the NIH (R01AR044031) and Canadian Institutes for Health Research (MOP-12080 and MOP-81288) to MAR. DC was an American Association of Anatomists Scholar, and this research was funded in part by the American Association of Anatomists. CFB was supported by the Swiss National Science Foundation.
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