Striated myogenesis and peristalsis in the fetal murine esophagus occur without cell migration or interstitial cells of Cajal

Abstract

Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal.

Fig. 1.

Sagittal sections of murine esophagi from E14.5 fetuses show progressive myogenin expression over 7 days of culture: day 0 (A), day 5 (B) and day 7 (C). a Myogenin expression (red nuclear staining) in the diaphragm. b Myogenin expression in 3–4 subpharyngeal nuclei. c Robust myogenin expression in the tongue. d Myogenin expression extending caudally along the rostral muscularis externa layers. e Myogenin expression in the muscularis externa approaching the stomach. Scale bars for large panels = 500 μm; scale bars for insets = 100 μm.

Fig. 2.

Mechanical disruption of the esophageal muscularis externa results in discontinuity of smooth muscle fluorescence. A Fresh whole mount E14.5 esophagus explanted from SMCG2 transgenic mice (which express eGFP in smooth muscle) showing 3 adjacent zones of muscularis externa disruption (arrows). B Fluorescent imaging shows loss of smooth muscle fluorescence through the disrupted zones (arrows).

Fig. 3.

Striated myogenesis occurs in esophagi with mechanically disrupted muscularis externa. A Sagittal section of E14.5 esophagus with a disrupted muscularis externa (arrow) but contiguity of the rostral and caudal segments after 7 days of culture shows myogenin expression on both sides of the zone of disruption (insets). The apparent dissociation to the right of the arrow (rostral) is a sectioning artifact, where a section of esophagus was positioned outside of the sectioning plane. B Myogenin expression is visible only in the subpharyngeal segment in a completely transected E14.5 esophagus after 7 days of culture, but is absent in the caudal segment (insets). Scale bars for large panels = 500 μm; scale bars for insets = 100 μm.

Fig. 4.

Smooth muscle maintains smooth muscle actin expression in culture for 7 days, but is attenuated by FGF-2. A Sagittal section of E14.5 esophagus after 7 days of culture shows robust expression of smooth muscle actin in all 3 smooth muscle layers of the esophagus (a = muscularis mucosa, b = muscularis externa inner layer, c = muscularis externa outer layer). B Smooth muscle actin expression is attenuated after 7 days of culture when FGF-2 is added to the culture media (a muscularis mucosa, b muscularis externa inner layer, c muscularis externa outer layer). Note the loss of actin in the outer gastric muscle layers. C Myogenin expression remains unaffected by culture in FGF-2. Scale bars = 500 μm. Inset PD98059 inhibits ERK1/2 phosphorylation by FGF-2. E14.5 esophageal lysates, immunoblotted with anti-phospho-ERK1/2 or anti-pan-ERK antibodies, show complete inhibition of ERK1/2 phosphorylation by FGF-2 after concurrent treatment with MEK inhibitor PD98059 (upper panel, lanes 3 and 4). Esophagi treated with FGF-2 alone show robust phosphorylation of ERK1 and ERK2 (upper panel, lanes 1 and 2). Total ERK expression is similar for all samples (lower panel).

Fig. 5.

ACK2 antibody penetrates esophagi in culture but fails to inhibit peristalsis. Sagittal section of E14.5 esophagus labeled with a biotinylated goat anti-rat antibody and detected with Texas Red-avidin. Fluorescence can be detected throughout the esophagus, indicating complete penetration of the antibody during the culture period.