Development of nicotinic receptor clusters and innervation accompanying the change in muscle phenotype in the mouse esophagus

Abstract

During development, the external muscle of the mouse esophagus undergoes a transdifferentiation from smooth to striated muscle (Patapoutian et al. [1995] Science 270:1818-1821). We now report on the development of the innervation accompanying the change in phenotype of the external muscle of the mouse esophagus. The phenotype of the muscle was monitored by using light and electron microscopy. Nicotinic acetylcholine receptors were localised by using a fluorescence conjugate of alpha-bungarotoxin, and neural elements were localised by using antisera to synaptophysin (a synaptic vesicle protein that was used to label all nerve terminals), the vesicular acetylcholine transporter (VAChT), calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), and vasoactive intestinal peptide (VIP). CGRP and VAChT were co-localised in the terminals of vagal motoneurons that innervate the external muscle, and NOS and VIP were co-localised in intrinsic (enteric) neurons, which provide some terminals that are associated with motor endplates. Cells exhibiting striations were first observed in the outer layers of the most rostral regions of the esophagus of embryonic day 15 (E15) mice. Clusters of nicotinic acetylcholine receptors were also first observed at the rostral end of the esophagus of E15 mice, and developed in a rostrocaudal progression that coincided with the appearance of striations within the muscle cells. Synaptophysin-, VAChT- and NOS-immunoreactive nerve terminals were present within the external muscle prior to the formation of receptor clusters, and their appearance did not follow any apparent rostrocaudal sequence. Surprisingly, not all of the receptor clusters at E15 had synaptophysin- and VAChT-immunoreactive nerve terminals closely associated with them. However, from E18 on, almost all of the clusters had synaptophysin-immunoreactive nerve terminals in close association. At late embryonic and early postnatal stages, there was a rostrocaudal gradient in the proportion of receptor clusters having VAChT-immunoreactive nerve terminals associated with them. Nerve terminals associated with nicotinic receptor clusters did not show detectable CGRP-immunoreactivity until one to two weeks after the appearance of synaptophysin- and VAChT-immunoreactivity. The NOS-immunoreactive neurons did not show detectable VIP-immunoreactivity until three days after NOS could be detected. These results show that the appearance of clusters of nicotinic receptors in the external muscle of the esophagus coincides with the expression of a striated muscle phenotype, but not with the presence of ingrowing nerve terminals. However, many of the receptor clusters that were observed first were apparently uninnervated.

Evidence for the involvement of neurotrophins in muscle transdifferentiation and acetylcholine receptor transformation in the esophagus of Myf5−/−:MyoD−/− and NT‐3−/− embryos

“…Previous results have shown that esophageal SM cells fail to transdifferentiate into striated muscle in Myf5 -/-:MyoD -/-embryos, even though this finding appears to occur in the wild-type esophagus (Patapoutian et al, 1995;Sang and Young, 1997;Kablar et al, 2000). Furthermore, SM cells in the esophagus have been shown to contain subtypes M1-M5 of mAChR (Wang et al, 2000), whereas subtype M2 has been identified on the esophageal enteric nerve terminals (Neuhuber et al, 2001).…”

“…Furthermore, SM cells in the esophagus have been shown to contain subtypes M1-M5 of mAChR (Wang et al, 2000), whereas subtype M2 has been identified on the esophageal enteric nerve terminals (Neuhuber et al, 2001). By contrast, the appearance of nAChR clusters coincides with the expression of a striated muscle phenotype (Sang and Young, 1997). To determine whether SM cells of the double-mutant esophagus nonetheless undergo a transition from mAChRs to nAChRs, FITC-␣-bungarotoxin staining and antinAChR immunohistochemistry was performed on relevant embryonic stages (i.e., E15.5-E18.5).…”

“…Collectively, our data strongly suggest a role for neurotrophins (especially NT-3) and their receptors in the esophageal muscle transdifferentiation and in the acetylcholine receptor type transformation. (Sang and Young, 1997) (Fig. 1).…”

“…At embryonic day (E) 15.5, skeletal muscle begins to form from the SM cells as an example of transdifferentiation (i.e., the change in phenotype of a differentiated cell; Patapoutian et al, 1995;Sang and Young, 1997;Kablar et al, 2000). At postnatal day (P) 14, the esophagus has reached its adult phenotype, consisting of an external layer that has almost completely transformed into striated muscle (Sang and Young, 1997). This entire process has been shown to depend on the primary myogenic regulatory factors (MRFs) Myf5 and MyoD, with Myf5 allowing for the initiation of the process at E15.5 or MyoD (in Myf5 -/-embryos) by E17.5 (Kablar et al, 2000).…”

“…The pri-mary focus of this study is to determine whether the esophageal innervation and neurotrophic factors have a role in the change of the muscle phenotype and the transition in the acetylcholine receptor type from muscarinic to nicotinic (Sang and Young, 1997). Esophageal motor innervation is quite complex and can be described as originating from both the central nervous system (CNS) as well as the ENS (enteric nervous system; Young, 1997, 1998;Neuhuber et al, 2001;Wö rl et al, 2002).…”