Innervation of the Esophagus in MiceThat Lack MASH1

Q. SANG,1 D. CIAMPOLI,1 U. GREFERATH,2 L. SOMMER,3 AND H.M. YOUNG1*1Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3052,

Victoria,Australia2Walter and Eliza Hall Institute, Parkville 3052, Victoria, Australia

3Institute of Cell Biology, Swiss Federal Institute of Technology,CH-8093 Zurich, Switzerland

ABSTRACTThe striated muscle of the esophagus differs from other striated muscle, because it

develops by the transdifferentiation of smooth muscle, and the motor end plates receive a dualinnervation from vagal (cholinergic) motor neurons and nitric oxide synthase (NOS)-containing enteric neurons. Mash1 2/2 mice have no enteric neurons in their esophagus anddie within 48 hours of birth without milk in their stomachs (Guillemot et al. [1993] Cell75:463–476). In this study, the innervation of the esophagus of newborn Mash1 2/2, Mash11/2 and wild type mice was examined. There was no difference between Mash1 2/2, Mash11/2, and wild type mice in the transdifferentiation of the muscle and the development ofnicotinic receptor clusters. However, there were significantly more cholinergic nerve termi-nals per motor end plate in Mash1 2/2 mice than Mash1 1/2 or wild type mice. Each of theMash1 2/2 mice had fewer than 50 NOS neurons per esophagus, compared with approxi-mately 3,000 in wild type mice. Newborn Mash1 1/2 mice also contained significantly fewerNOS neurons than wild type mice. In Mash1 2/2 mice, NOS nerve fibers were virtuallyabsent from the external muscle but were present at the myenteric plexus. Unlike that ofnewborn wild type mice, the lower esophageal sphincter of Mash1 2/2 mice lacked NOS nervefibers; this may explain the absence of milk in the stomach. We conclude that 1) thetransdifferentiation of the esophageal muscle and the development of the extrinsic innerva-tion do not require enteric neurons or MASH1, 2) extrinsic NOS neurons only innervate themyenteric plexus. J. Comp. Neurol. 408:1–10, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: enteric; nitric oxide synthase; vagus; lower esophageal sphincter;

transdifferentiation

In humans, the external muscle of the upper one-third ofthe esophagus is composed of striated muscle; in the ratand mouse, the external muscle of almost the entireesophagus consists of striated muscle. In all mammals inwhich it has been investigated, the striated muscle of theesophagus is innervated by cholinergic neurons that arisein the nucleus ambiguus and project to the esophagusthrough the vagus nerves (see Cunningham and Saw-chenko, 1990). Peristaltic movements in the striated muscleportion of the esophagus are therefore controlled directlyby the brainstem. There are also enteric neurons betweenthe layers of striated muscle in the mammalian esopha-gus, but their role is unknown. In addition to the choliner-gic nerve terminals innervating the striated muscle, thereare nitric oxide synthase (NOS)-containing nerve termi-nals at many motor end plates in the mouse and ratesophagus (Neuhuber et al., 1994; Worl et al., 1994, 1997;Sang and Young, 1997). Because there are many enteric

neurons containing NOS within the esophagus, it has beensuggested that NOS nerve terminals within the striatedmuscle arise from enteric neurons (Neuhuber et al., 1994),although an extrinsic origin has not been ruled out.

Unlike skeletal striated muscle, which develops frommyoblasts and myotubes, the striated muscle of the mouseesophagus develops by the transdifferentiation of differen-tiated smooth muscle during late embryonic and earlypostnatal development (Patapoutian et al., 1995). Thetransdifferentiation starts at the rostral end of the esopha-

Grant sponsor: NHMRC (Australia).*Correspondence to: Dr. H.M. Young, Department of Anatomy & Cell

Biology, University of Melbourne, Parkville 3052, Victoria, Australia.E-mail: h.young@anatomy.unimelb.edu.au

Received 18 September 1998; Revised 7 December 1998; Accepted 10December 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 408:1–10 (1999)

r 1999 WILEY-LISS, INC.

gus at embryonic day 16 (E16) and proceeds rostrocau-dally; striated muscle is present along the entire esopha-gus by postnatal day 8 (P8; Patapoutian et al., 1995).Although cholinergic nerve terminals are present in theexternal muscle while the muscle is still phenotypicallysmooth muscle, nicotinic receptor clusters do not appearuntil the muscle begins to show a striated muscle pheno-type (Sang and Young, 1997). Thus, the appearance ofnicotinic receptor clusters occurs rostrocaudally, coincid-ing with the appearance of striated muscle. The factorsthat influence the transdifferentiation from smooth tostriated muscle have yet to be determined.

Like the external muscle, the enteric nervous system ofthe mouse esophagus shows unusual developmental fea-tures. The neurons and glial cells of the enteric nervoussystem are derived from the neural crest. However, theenteric nervous system of esophagus differs from that inother parts of the gastrointestinal tract in the level of theneural axis from which it arises and in the genes requiredfor its development. The vast majority of enteric neuronsin the stomach and intestine arise from ‘‘vagal’’ level(somites 1–7) neural crest (Yntema and Hammond, 1954;Le Douarin and Teillet, 1973; Epstein et al., 1994) andmigrate rostrocaudally through the gastrointestinal tract(Kapur et al., 1992; Young et al., 1998). However, most ofthe enteric neurons in the esophagus do not arise fromvagal level neural crest (Epstein et al., 1994), and itappears that they arise from anterior trunk level neuralcrest, which also gives rise to the superior cervical gan-glion (Durbec et al., 1996). Mice in which the genesencoding glial-derived neurotrophic factor (GDNF) or itsreceptor, c-RET, have been inactivated have dramaticallyreduced numbers or an absence of enteric neurons in theirstomachs and an absence of neurons in the gastrointesti-nal tract caudal to the stomach; however, enteric neuronsare present in the esophagus (Schuchardt et al., 1994;Durbec et al., 1996; Moore et al., 1996; Pichel et al., 1996;Sanchez et al., 1996). In contrast, mice in which the geneencoding the transcription factor, MASH1, has been inacti-vated, have no enteric neurons in their esophagus; how-ever, enteric neurons are present in their stomachs andintestine, but in reduced numbers (Guillemot et al., 1993;Blaugrund et al., 1996). Mash1 2/2, gdnf 2/2, and Ret2/2 mice all die within 24 hours of birth, and, whereasthere is no milk in the stomach of Mash1 2/2 mice even6–12 hours after birth (Guillemot et al., 1993), milk is presentin the stomach of gdnf 2/2 and Ret 2/2 mice, but it fails toprogress to the small intestine (Schuchardt et al., 1994).

Because Mash1 2/2 mice lack enteric neurons in theiresophagus, they provide an opportunity to determinewhether the presence of enteric neurons is necessary forthe transdifferentiation of the external muscle to occur andto examine aspects of the interactions between the extrin-sic and intrinsic innervation of the esophagus. Thus, theaims of the current study were to examine the esophagusof newborn Mash1 2/2 mice 1) to determine whether thetransdifferentiation of the external muscle and the devel-opment of nicotinic acetylcholine receptors require thepresence of enteric neurons, and 2) to examine possiblecauses of the absence of milk in the stomach of newbornMash1 2/2 mice. Esophageal peristalsis in the mouse isprobably controlled directly by the brainstem (see above).However, in order for a bolus of food to enter the stomach,there also must be a reflex relaxation of the lower esophagealsphincter. We therefore examined both the extrinsic innerva-

tion of the esophagus and the innervation of the lower esopha-geal sphincter of newborn Mash1 2/2 mice and comparedthem with their Mash 1/1 and Mash1 1/2 litter mates.

MATERIALS AND METHODS

All of the experiments were performed on the newbornoffspring (12 hours old or less) of matings of Mash1 1/2mice that were obtained originally from the HowardHughes Medical Institute, California Institute of Technol-ogy. All of the protocols were approved the by the AnimalEthics Committee of the University of Melbourne. Themice were killed by decapitation; the esophagus was thenremoved and processed for anatomical studies, and theliver was removed and frozen for later genotyping by usinga polymerase chain reaction (PCR). All of the anatomicstudies were done without knowing the genotype of themice.

Analysis of the innervation of the esophagus

The esophagus from most of the newborn mice wasopened, the mucosa were pinned down and exposed tofluorescein-isothiocyanate-conjugated a-bungarotoxin(FITC-a-BTX), fixed, and then processed for immunohisto-chemistry as described previously (Sang and Young, 1997).The primary antisera used were a goat antivesicularacetylcholine transporter (VAChT; 1:1,000; Chemicon Inter-national Inc., Temecula, CA), a sheep antineuronal nitricoxide synthase (NOS; 1:2,000; Young and Ciampoli, 1998)or a rabbit antineuronal NOS (1:1,000; Young et al., 1997),and the secondary antisera used were a biotinylateddonkey anti-sheep (1:100; Jackson ImmunoResearch, WestGrove, PA) followed by streptavidin Texas Red (1:100;Amersham, Melbourne, Australia) or a donkey anti-rabbitFITC (1:50; Amersham). To determine the percentage ofFITC-a-BTX-labelled receptor clusters that had VAChT-immunoreactive nerve varicosities associated with them,the first 50 FITC-a-BTX-labelled receptor clusters encoun-tered at 2-mm intervals along the entire esophagus wereexamined by using a 3100 objective lens. For thosereceptor clusters with varicosities associated with them inthe rostral 4 mm of the esophagus, the number of VAChT-immunoreactive nerve terminals at each receptor clusterwas also determined from video images of each receptorcluster. Some preparations were processed for NOS immu-nohistochemistry, so that the number of NOS-containingneurons in the myenteric plexus of each preparation couldbe determined. If there were fewer than 100 NOS-immunoreactive cell bodies along the entire esophagus,then each stained neuron was counted. If there were morethan 100, then the number of NOS-immunoreactive neu-rons in randomly sampled areas along the esophagustotaling 2–3 mm2 was counted. The total number of NOSneurons in each esophagus was then estimated afterdetermining the surface area. The innervation of the loweresophageal sphincter was examined by processing frozensections through the most caudal regions of the esophagusfor NOS and VAChT immunohistochemistry. Segments ofesophagus from the rostral and caudal ends of someanimals were embedded in resin, and 0.5–1.0 µm sectionswere cut, so that the phenotype of the muscle cells could beascertained. Images were obtained by using an Image-Point cooled CCD camera (Photometrics Ltd., Tucson, AZ)and V for Windows imaging software (Digital Optics Ltd.,Auckland, New Zealand). Each image was processed by

2 Q. SANG ET AL.

using a sharpened filter and contrast adjustment. Plateswere made by using Corel PhotoPaint and Draw software(Corel Corp., Dublin, Ireland).

Genotyping of newborn mice

PCR was used to identify the genotype of the newbornmice. The liver was incubated overnight at 55°C in 700 µlof buffer (50 mM Tris-HCl, 100 mM NaCl, 100 mMethylenediamine tetraacetic acid, and 1% weight/volumesodium dodecyl sulfate) plus 20 µl of 20 mg/ml proteinaseK, and DNA was extracted using by phenol/chloroform-isoamyalcohol followed by chloroform isoamylalcohol. PCRwas performed by using the primers and conditions de-scribed by Blaugrund et al. (1996).

RESULTS

All of the experiments were performed on newborn mice(less than 12 hours old) that resulted from intercrossesbetween Mash1 1/2 parents. The mice were genotyped(Fig. 1) after the nicotinic acetylcholine receptors andinnervation had been examined anatomically. The pres-ence or absence of milk in the stomachs was also noted. Nomilk was found in the stomach of any mouse that was latergenotyped as Mash1 2/2, as reported previously (Guille-mot et al., 1993). However, milk was also absent from thestomachs of many of the newborn Mash1 1/1 and Mash11/2 mice, because, in most cases, the mice were removedfrom their mothers as soon as possible after birth.

Presence of striated muscle and developmentof nicotinic receptor clusters

At birth, the external muscle of the rostral two-thirds ofthe esophagus of newborn mice has transdifferentiatedinto striated muscle, and the caudal one-third is stillsmooth muscle (Patapoutian et al., 1995). Similar to thefindings in wild type mice, striated muscle was present inthe rostral part of the esophagus of Mash1 2/2 mice (Fig.2A,B), whereas the muscle in the caudal region did notexhibit striations. FITC-a-BTX-labelled nicotinic receptorclusters first appear in the external muscle of the mostrostral part of the esophagus of E15 wild type mice andspread rostrocaudally, so that receptor clusters are presentalong the rostral two-thirds of the esophagus at birth andalong the entire esophagus by P7 (Sang and Young, 1997).

The esophagus of newborn mice is approximately 12 mmlong, and, in Mash1 1/1, Mash1 1/2, and Mash1 2/2mice, nicotinic receptor clusters were present in the rostraltwo-thirds of the esophagus (Fig. 2C,D).

Presence of cholinergic(VAChT-immunoreactive) nerve terminalsat FITC-a-BTX-labelled receptor clusters

VAChT-immunoreactive nerve terminals were associ-ated with many of the FITC-a-BTX-labelled nicotinicreceptor clusters in the external muscle of Mash1 1/1,Mash1 1/2, and Mash1 2/2 mice, with no qualitativedifferences between the different genotypes (Fig. 2E,F).Not all of the receptor clusters had nerve terminalsassociated with them. Quantitative examination showedthat the proportion of receptor clusters that had VAChT-immunoreactive nerve terminals associated with them inMash1 2/2 mice was similar to that seen in wild type andheterozygote mice (Fig. 3). However, the number of VAChT-positive nerve varicosities at each receptor cluster ofMash1 2/2 mice was significantly higher than that inMash1 1/2 or Mash1 1/1 mice (Fig. 4).

NOS-immunoreactive neurons

In newborn wild type mice, NOS-immunoreactive cell bod-ies were present in the myenteric plexus along the entireesophagus, and NOS-immunoreactive nerve terminals werepresent within the myenteric plexus and the external muscle(Fig. 5A,E). The total number of NOS-immunoreactive cellbodies in the myenteric plexus of the entire esophagus ofMash1 1/1 mice was significantly higher than that in Mash11/2 mice (Fig. 6). However, the distribution of NOS-immuno-reactive cell bodies and nerve terminals within the myentericplexus and external muscle of Mash1 1/2 mice appeared to beidentical to that in wild type mice. Mash1 2/2 mice have beenreported to have no enteric neurons in their esophagus (Guille-mot et al., 1993). In the current study, NOS-immunoreactivecell bodies were observed only very rarely along the esophagusof Mash1 2/2 mice (Fig. 5C). Of nine newborn Mash1 2/2mice in which the total number of NOS neurons was deter-mined, between 2 and 48 NOS-positive neurons were observed(Fig. 6), and they were usually concentrated at either the mostrostral or the most caudal region of the esophagus. A smallnumber of NOS-immunoreactive cell bodies were observedcommonly along the vagal trunks of Mash1 2/2 mice (Fig.5D), whereas they were uncommon in wild type mice. NOS-immunoreactive nerve fibers were present in the vagal nervesof all mice. Most of the Mash1 2/2 mice did not possess anyNOS-positive nerve terminals within the external muscle,including the motor end plates; extremely sparse, NOS-positive nerve fibers that ran parallel to the muscle layerswere observed in about one-third of the mutant mice (Fig. 5F).However, in all Mash1 2/2 mice examined, there was a plexusof NOS-immunoreactive nerve terminals present between thetwo layers of external muscle, where the myenteric plexuswould normally be located (Fig. 5B).

Although NOS neurons comprise the majority of intrin-sic neurons in the mouse esophagus, there are also otherneurochemical types of neurons present (Sang and Young,1998). To determine whether there is a specific decrease inthe number of NOS neurons or whether there is a decreasein the total number of enteric neurons in the esophagus ofMash1 1/2 mice, wholemount preparations were pro-cessed for immunohistochemistry by using antibodies tothe panneuronal markers, neuronal specific enolase and

Fig. 1. Polymerase chain reaction (PCR) analysis of three newbornprogeny from a Mash1 1/2 intercross. The 270-base-pair (bp) amplifi-cation product is generated from the neo mutant allele, whereas the468-bp amplification is generated only from the Mash1 wild typeallele. Lane 1 is from a homozygous mutant (Mash1 2/2), lane 2 isfrom a heterozygous mouse (Mash1 1/2) and lane 3 from a wild typemouse (1/1) mouse.

ESOPHAGUS INNERVATION IN MICE LACKING MASH1 3

PGP9.5. However, we were unable to obtain consistent(nonpatchy) staining along the esophagus with either ofthese antibodies. Thus, accurate counts of the total num-ber of neurons could not be made.

Innervation of the lower esophagealsphincter

The lower esophageal sphincter of newborn mice con-sisted of a thick layer of smooth muscle at the junction of

the esophagus and the stomach, which was best visualizedin longitudinal sections (parallel to the length of theesophagus). In newborn wild type and Mash1 1/2 mice,NOS-immunoreactive cell bodies and nerve terminalswere present in the myenteric plexus underneath thesphincter, and numerous NOS-immunoreactive nerve ter-minals were present within the circular muscle (Fig. 7A).VAChT-immunoreactive nerve terminals also were ob-served in the myenteric plexus and circular muscle, but

Fig. 2. A,B: Resin sections (0.5 µm thick) of the rostral region of theesophagus of a wild type mouse (A) and a Mash1 2/2 mouse (B).Striations (arrows) are present in cells in the external muscle (extmuscle)layers of mice of both genotypes. A neuron (asterisk) is presentbetween the two muscle layers in the wild type mouse (A), but theregion between the two muscle layers (asterisks) is devoid of neuronsin the mutant (B). C,D: Low-power image of fluorescein-isothiocyanate-

conjugated a-bungarotoxin (FITC-a-BTX)-labelled nicotinic receptorclusters in the external muscle of a wild type mouse (C) and a Mash12/2 mouse (D). E,F: Vesicular acetylcholine transporter (VAChT)-immunoreactive nerve varicosities (red) associated with an FITC-a-BTX-labelled receptor cluster (green) from a wild type mouse (E) and aMash1 2/2 mouse (F). Scale bars 5 10 µm in B (also applies to A) andC (also applies to D), 2 µm in F (also applies to F).

4 Q. SANG ET AL.

they were very sparse (Fig. 7C). Double-label experimentsshowed that there was no overlap between NOS andVAChT in any nerve terminals within the sphincter. Innewborn Mash1 2/2 mice, NOS-immunoreactive cell bod-ies were sometimes observed in the myenteric plexus, butNOS nerve terminals were observed only rarely in themuscle forming the sphincter (Fig. 7B). However, VAChT-immunoreactive nerve terminals were present within thesphincter muscle of Mash1 2/2 mice (Fig. 7D).

DISCUSSION

Presence of extrinsic nerves in theesophagus of Mash1 2/2 mice

The cholinergic nerve terminals present at nicotinicreceptor clusters in the external muscle of the mouseesophagus arise from the nucleus ambiguus (Sang andYoung, 1998). Retrograde and anterograde tracing studiesof embryonic rats (Rinaman and Levitt, 1993) and mice

Fig. 3. The proportion of FITC-a-BTX-labelled nicotinic receptorclusters at different rostrocaudal levels that have VAChT-immunoreac-tive nerve terminals associated with them in newborn wild type mice(n 5 5), in Mash1 1/2 mice (n 5 6), and in Mash1 2/2 mice (n 5 5).

Values represent means 6 S.E.M. There was no significant differencebetween the three genotypes (Mann-Whitney test; P , 0.05). At eachrostrocaudal level of each mouse, 20 receptor clusters were counted.

Fig. 4. Number of VAChT-immunoreactive nerve varicosities ateach FITC-a-BTX-labelled receptor cluster in newborn Mash1 2/2,Mash1 1/2, and wild type mice. Values represent means 6 S.E.M.There were significantly more VAChT-immunoreactive varicosities

per receptor cluster in Mash1 2/2 mice than in Mash1 1/2 or wildtype mice, but there was no significant difference between Mash1 +/2 and wild type mice (Mann-Whitney test).

ESOPHAGUS INNERVATION IN MICE LACKING MASH1 5

(Sang and Young, 1998) have shown that the vagal innerva-tion of the esophagus develops during midembryonic stages.In the present study, we have shown that cholinergic(VAChT-immunoreactive) nerve terminals were present inassociation with nicotinic receptor clusters in newbornMash1 2/2 mice. In newborn wild type mice, not all of thereceptor clusters have cholinergic nerve terminals associ-ated with them, and the proportion of innervated receptorclusters decreases rostrocaudally. There was no differencebetween wild type, Mash1 1/2 and Mash1 2/2 mice in the

proportion of receptor clusters innervated by cholinergicnerve terminals. Thus, in most respects, the cholinergicinnervation of the external muscle arising from the nucleusambiguus appears to develop normally in Mash1 2/2mice. It is noteworthy that there were significantly morecholinergic nerve varicosities per motor end plate inMash1 2/2 mice than in Mash1 1/2 or wild type mice.One possible reason for the increased number of choliner-gic varicosities in Mash1 2/2 mice is that NOS nerveterminals are absent from the external muscle, whereas,

Fig. 5. Wholemount preparations of external muscle from newbornmice processed for nitric oxide synthase (NOS) immunohistochemis-try. A: NOS-immunoreactive cell bodies and nerve terminals in themyenteric plexus (MP) of a wild type mouse. B: NOS-immunoreactivenerve terminals in a plexus between the muscle layers (MP) in aMash1 2/2 mouse. C: A single NOS-immunoreactive neuron in the

myenteric plexus (MP) of a Mash1 2/2 mouse. D: Two NOS neuronsalong the vagal nerve trunk of a Mash1 2/2 mouse. E: NOS-immunoreactive nerve fibers (arrows) in the external muscle of a wildtype mouse. F: An NOS nerve fiber (arrow) in the external muscle of aMash1 2/2 mouse. Such fibers were very rare. Scale bars 5 50 µm inA–E, 25 µm in F.

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in wild type mice, NOS nerve terminals are present atmotor end plates along with cholinergic nerve terminals(Sang and Young, 1997). It is feasible that, in wild typemice, the presence of NOS nerve terminals in some waylimits the number of cholinergic nerve terminals presentat each motor end plate. In ls/ls mice (Payette et al., 1987)and in humans suffering from Hirschsprung’s disease(Smith, 1967; Gannon et al., 1969; Kapur, 1993), there isalso a hyperinnervation of the aganglionic region of bowelby nerve fibers of extrinsic origin. Thus, in the gastrointes-tinal tract, intrinsic enteric neurons may influence theextent of the innervation from extrinsic neurons.

Dissecting the contribution of intrinsic neurons to theinnervation of the esophagus from that of extrinsic neu-rons has proved difficult. Because Mash1 2/2 mice largelylack esophageal enteric neurons, they provide an opportu-nity to examine the targets of extrinsic neurons. In new-born Mash1 2/2 mice, although NOS-containing nervefibers were extremely sparse in the external muscle (mostmutants had none), a plexus of NOS-immunoreactivenerve terminals was present between the two layers ofstriated muscle, where the myenteric plexus is found inwild type mice. Because there were very few intrinsic NOSneurons in the myenteric plexus of Mash1 2/2 mice, mostof the NOS nerve terminals at the level of the myentericplexus must arise extrinsically. A recent retrograde studyhas shown that the major extrinsic source of NOS nerveterminals to the mouse esophagus is the dorsal motornucleus of the vagus (Sang and Young, 1998). The absenceof NOS nerve terminals from the external muscle of Mash12/2 mice suggests that all of the NOS nerve terminalswithin the external muscle of wild type mice arise fromintrinsic neurons, as suggested previously (Neuhuber etal., 1994), and that the extrinsic NOS neurons innervatethe myenteric plexus but not the external muscle.

Absence of enteric innervation of theesophagus in newborn Mash1 2/2 mice

The absence of intrinsic neurons from the esophagus ofMash1 2/2 mice has been reported previously (Guillemotet al., 1993). A large proportion of enteric neurons in theesophagus of the mouse contains NOS (Sang and Young,1998). We found, as expected, that there were very fewintrinsic NOS neurons (,1% of the number in wild typemice) in the esophagus of Mash1 2/2 mice. In addition,the total number of NOS neurons in the esophagus ofMash1 1/2 mice was found to be significantly lower thanthat in newborn wild type mice. The decrease in thenumber of NOS neurons in Mash1 1/2 and Mash1 2/2mice may be due to a decrease in the number of neuralcrest cells migrating into the esophagus, to decreasedsurvival of neural crest-derived cells within the esopha-gus, to decreased numbers of neural precursors differenti-ating into neurons, or to a combination of these causes. Anumber of studies have shown that MASH1 appears to beessential for promoting the differentiation of presumablycommitted neural precursors (Guillemot et al., 1993; Som-mer et al., 1995). For example, although Mash1 2/2 micelack anterior sympathetic chain ganglia, neural crest cells(labelled by c-ret probes) migrate to the correct locationsadjacent to the dorsal aorta but fail to differentiate intoneurons (Guillemot et al., 1993; Sommer et al., 1995).Therefore, it seems most likely that, in the esophagus ofMash1 2/2 mice (and, to a lesser extent, Mash1 1/2mice), decreased numbers of NOS enteric neuron precur-sors differentiate into neurons. In Mash1 1/2 mice, itremains to be determined whether there is an overalldecrease in the number of enteric neurons in the esopha-gus or whether there is a selective decrease in the numberof NOS neurons.

Fig. 6. Total number of NOS-immunoreactive neurons in the entire esophagus of newborn mice. Thenumber of NOS neurons in Mash1 2/2 mice is significantly less than that in Mash1 1/2 or wild typemice. The number of NOS cells in Mash1 1/2 mice is also significantly less than that in wild type mice(Mann-Whitney test).

ESOPHAGUS INNERVATION IN MICE LACKING MASH1 7

Innervation of the lower esophagealsphincter in Mash1 2/2 mice

In a range of species, the muscle of the lower esophagealsphincter is innervated by cholinergic nerve terminalsthat excite the muscle and thus promote closure of thesphincter and NOS nerve fibers, which are responsible forthe relaxation (through the release of nitric oxide) of thesphincter upon the arrival of a bolus of food (Tottrup et al.,1991; Murray et al., 1991; Yamato et al., 1992; Sohn et al.,1993; Brookes et al., 1996; Ward et al., 1998). In thecurrent study, we found that the innervation of the sphinc-ter by cholinergic and NOS nerve terminals is alreadyestablished in newborn wild type mice. However, althoughthe cholinergic innervation of the sphincter of newbornMash1 2/2 mice appeared normal, there were almost noNOS nerve terminals. In the guinea pig, the NOS nervefibers in the lower esophageal sphincter arise from entericneurons that are located rostral to and within the sphinc-ter, whereas the cholinergic nerve fibers arise almostexclusively from enteric neurons that are located withinthe sphincter (Brookes et al., 1996). Because we observedsome intrinsic NOS neurons in the myenteric plexus at thelevel of the lower esophageal sphincter of Mash1 2/2 mice,it appears that the majority of the NOS innervation of the

sphincter in the mouse arises from enteric neurons rostralto the sphincter, which are absent in Mash1 2/2 mice.

Absence of milk from the stomachof Mash1 2/2 mice

None of the Mash1 2/2 mice examined in this study hadmilk in their stomachs, as reported previously (Guillemotet al., 1993). In adult mice, because the external muscle ofthe esophagus is composed exclusively of striated musclethat is innervated by neurons in the nucleus ambiguus,esophageal peristalsis is controlled directly by the brain-stem. The role of intrinsic enteric neurons in peristalsis inthe mouse esophagus is unknown, as is the role of entericneurons in the striated muscle portion of the esophagus ofspecies in which only the rostral half of the externalmuscle is striated muscle. In the striated muscle region ofthe esophagus, vagal motor neurons directly innervate themuscle, whereas, in the smooth muscle region, vagalneurons innervate esophageal enteric neurons, which, inturn, innervate the smooth muscle. The current study hasshown that the absence of milk from the stomach of Mash12/2 mice is not due to defects in the cholinergic (extrinsic)innervation of the striated muscle, because cholinergicnerve terminals and nicotinic receptor clusters were pre-

Fig. 7. Frozen longitudinal sections through the lower esophagealsphincter of newborn mice processed for NOS (A,B) or VAChT (C,D)immunohistochemistry. NOS-immunoreactive nerve terminals arepresent in the sphincter of the wild type mouse (A) but not the Mash12/2 mouse (B). Two NOS-immunoreactive neurons can be seen in thevagal trunk (arrowheads in B), and a single NOS neuron (arrow in B)

is present in the myenteric plexus in the section from the Mash1 2/2mouse. VAChT-immunoreactive nerve terminals in the sphincter of awild type mouse (C) and a Mash1 2/2 mouse (D). Sparse VAChT-immunoreactive nerve terminals are present in both types of mice. cm,Circular muscle; lm, longitudinal muscle; m, mucosa; mp, myentericplexus. Scale bars 5 50 µm.

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sent at motor end plates in Mash1 2/2 mice. However, inneonatal mice, only the rostral two-thirds of the externalmuscle has transdifferentiated into striated muscle, andthe external muscle in the caudal one-third of the esopha-gus is still smooth muscle (Patapoutian et al., 1995); thesmooth muscle presumably is innervated by intrinsicneurons. Because there are almost no intrinsic neurons inthe esophagus of Mash1 2/2 mice, it is possible that theabsence of milk in the stomach is due to an absence ofperistalsis in the caudal one-third of the esophagus. It isalso feasible that the presence of enteric neurons in thestriated muscle esophagus of adult animals is a develop-mental ‘‘hangover’’ from embryonic and neonatal stageswhen the external muscle was smooth muscle.

Another likely reason for an absence of milk from thestomach of Mash1 2/2 mice is the absence of NOS nervefibers from the lower esophageal sphincter. For ingestedfood to enter the stomach, the lower esophageal sphinctermust relax in response to the progression of a bolus of fooddown the esophagus. Because relaxation of the loweresophageal sphincter is mediated largely by nitric oxide(see above), it would seem that relaxation of the sphinctercould not occur in Mash1 2/2 mice. Mash1 2/2 mice,therefore, show characteristics similar to those of humanswith achalasia of the esophagus. Achalasia is a disorder inwhich the lower esophageal sphincter does not relax inresponse to the advance of a food bolus, and it is character-ized by a dearth of nerve fibers in the muscle of thesphincter; extrinsic nerve fibers do not appear to beaffected (Faussone-Pellegrini et al., 1985; Wattchow andCosta, 1996). Achalasia is primarily manifest in adults.Because MASH1 is expressed only transiently duringembryonic development (Lo et al., 1991) and appears to berequired for the differentiation of neuronal precursors(Sommer et al., 1995), it is very unlikely that defects inMASH1 function underlie achalasia of the esophagus.

Transdifferentiation of the external musclefrom smooth to striated muscle and the

development of nicotinic receptor clusters

In the current study, striated muscle and nicotinicreceptor clusters were found to be present in newbornMash1 2/2 mice with a distribution similar to that seen innewborn wild type mice. Thus, intrinsic neurons andMASH1 are not required for the transdifferentiation of themuscle or for the appearance and clustering of nicotinicreceptors on esophageal muscle.

Role of MASH1 in the developmentof enteric neurons

Enteric neurons are present in the stomach and intes-tine of Mash1 2/2 mice, although some subpopulations ofneurons appear to be missing (Guillemot et al., 1993;Blaugrund et al., 1996; Gershon, 1997, 1998). Some of thetypes of neurons present in the mouse esophagus aresimilar to those in other parts of the mouse gut in terms ofthe combinations of neurotransmitters that they contain(Sang and Young, 1996, 1998). For example, neuronscontaining NOS plus vasoactive intestinal peptide arepresent throughout the mouse gastrointestinal tract. Thus,the requirement for MASH1 by enteric neuron precursorsin the esophagus does not appear to be related to thepresence of neuronal types that are exclusive to theesophagus. Recent studies have shown that MASH1 in-duces the expression of another transcription factor,

Phox2a, which, in turn, induces the expression of c-RET(the receptor for GDNF; Hirsch et al., 1998; Lo et al., 1998).It is likely that most or all enteric neuron precursorsexpress MASH1 and c-RET (Lo et al., 1991; Pachnis et al.,1993); therefore, it remains to be determined why MASH1,but not c-RET, is necessary for the development of esopha-geal neurons and why c-RET, but not MASH1, is necessaryfor the development of neurons in the stomach and intes-tine.

ACKNOWLEDGMENTS

We thank Dr. David Anderson for supplying the breed-ing pairs of Mash1 1/2 mice and Professor John Furnessfor valuable comments on the paper.

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