REGULAR ARTICLE

C-kit-immunopositive interstitial cells of Cajal in humanembryonal and fetal oesophagus

Goran Radenkovic & Ivan Ilic & Dragoljub Zivanovic &

Slobodan Vlajkovic & Vladimir Petrovic &

Olivera Mitrovic

Received: 11 January 2010 /Accepted: 25 February 2010 /Published online: 30 April 2010# Springer-Verlag 2010

Abstract Interstitial cells of Cajal (ICC) are morphologi-cally and functionally intercalated between the elements ofthe enteric nervous system and the smooth muscle cells(SMCs) in the musculature of the digestive tract. Kitimmunohistochemistry reliably identifies the location ofthese cells and provides information on changes in ICCdistribution and density. Human oesophagus specimens (7embryos, 23 fetuses at 7-27 weeks gestational age; bothsexes) were exposed to Kit antibodies to determine ICCdifferentiation. Enteric plexuses were examined immuno-histochemically by using anti-neuron-specific enolase,whereas the differentiation of SMCs was studied withantibodies against α-smooth-muscle actin and desmin. Byweek 7, c-kit-immunopositive cells were present along theentire oesophagus in the form of an uninterrupted layer

around the myenteric plexus (MP) elements. From thebeginning of the 3rd month, the number of ICC progres-sively decreased around the MP ganglia but increasedwithin the muscle layers. Concomitantly, differences in thenumber and distribution of ICC were established in thevarious portions of the oesophagus: specifically, ICC wereabundant in the lower portion, less numerous in the middleregion and rare in the upper part. By the 5th month ofdevelopment, the relationship as found in later develop-mental stages had been established: C-kit IR ICC werepresent within the circular muscle layer, within thelongitudinal layer and in the connective septa surroundingthe muscle bundles but were completely missing around theMP ganglia.

Keywords Interstitial cells of Cajal . Development .

Immunohistochemistry . Oesophagus .

Digestive tract . Human

Introduction

Interstitial cells of Cajal (ICC) are a distinct and specialisedcell population in humans and are distributed within thetunica muscularis of the digestive tract from the uppersphincter of the oesophagus (Faussone-Pellegrini andCortesini 1985b; Torihashi et al. 1999) to the innersphincter region of the anus (Hagger et al. 1998). Asubpopulation of ICC is considered to be the pacemakercells that generate electric slow waves responsible forperistaltic activity (Torihashi et al. 1995; Huizinga et al.1995). Some other populations of ICC are mediators ofenteric motor neurotransmission (Burns et al. 1996; Wardand Sanders 2001; Beckett et al. 2003), play a role inafferent neural signalling and act as stretch receptors

G. Radenkovic (*) :V. PetrovicDepartment of Histology and Embryology, Faculty of Medicine,University of Nis,Zoran Djindjic Blv 81,18000 Nis, Serbiae-mail: radenkog@gmail.com

I. IlicDepartment of Pathology, Faculty of Medicine, University of Nis,Nis, Serbia

D. ZivanovicPediatric Surgery and Orthopaedic Clinic of Nis Clinical Centre,Nis, Serbia

S. VlajkovicDepartment of Anatomy, Faculty of Medicine, University of Nis,Nis, Serbia

O. MitrovicDepartment of Histology and Pathology,Institute for Medical Research,Belgrade, Serbia

Cell Tissue Res (2010) 340:427–436DOI 10.1007/s00441-010-0957-9

(Thuneberg and Peters 2001; Suzuki et al. 2003). ICC arearranged in two- or three-dimensional networks of electri-cally connected cells that form close contacts with the nerveplexus and smooth muscle cells (SMCs; Belzer et al. 2002;Mitsui and Komuro 2002; Beckett et al. 2005).

ICC express the gene product of c-kit, a proto-oncogenethat encodes the receptor tyrosine kinase, Kit (Ward et al.1994; Huizinga et al. 1995). Labelling of Kit receptors orc-kit mRNA has provided an efficient means of identifyingICC at the light-mickroscopic level in a variety ofpreparations, including human specimens (Maeda et al.1992). A cytokine, termed steel factor or stem cell factor(SCF), has been identified as a c-kit ligand (Williams et al.1990).

The muscularis propria is responsible for the motorfunction of the human oesophagus. The oesophageal wall iscomposed of striated muscle in the upper part, smoothmuscle in the lower part and a mixture of the two in themiddle part (Kallmunzer et al. 2008). The myenteric plexus(MP) is well developed in smooth muscle but is alsopresent in the striated muscle part of the oesophagus (Kuoand Urma 2006).

The distribution and appearance of c-kit-immunopositivecells in the oesophagus have been previously examined inthe mouse (Rumessen et al. 2001), cat (Huizinga et al.2008), pig (Wu et al. 2003), rat (Farre et al. 2007), guineapig (Burns et al. 1997) and human (Faussone-Pellegrini andCortesini 1985b; Torihashi et al. 1999). The cat oesophagushas been used extensively as a model for the humanoesophagus (Blank et al. 1989; Preiksaitis and Diamant1999), since it is one of the few animal models with a largesmooth muscle component in the oesophageal musculature(Diamant 1997). ICC show different distribution patternsand morphological features depending on their anatomicallocation, according to which the ICC are classified intoseveral subtypes: ICC of the circular muscle (ICC-CM) arelocated in the circular muscle layer and within the loweroesophageal sphincter, ICC of the longitudinal muscle(ICC-LM) occur within the longitudinal layer and ICC ofthe septa (ICC-SEP) lie in the connective tissue septa thatsurround bundles of the muscle, whereas ICC do notaggregate around the MP (ICC-MY) or at the submucosalborder (ICC-SM), as in the small and large intestine(Torihashi et al. 1999; Hanani et al. 2005; Komuro 2006).

Reports on the presence of ICCs in the oesophagealstriated muscle portion are limited (Burns et al. 1997;Rumessen et al. 2001) and the functional significance ofICCs adjacent to the striated muscle fibres is still unclear.Little information is available regarding the early difer-entiation of these cells in the human oesophagus.

At the beginning of week 4, the neural crest cells enterthe foregut, migrate rostrocaudally to reach the terminalhindgut by week 7 and give rise to the MP (Fu et al. 2004).

Smooth muscle differentiation begins after the neural crestcells colonise the gut and mature along the rostrocaudalaxis (Wallace and Burns 2005). The ICC emerge from thegut mesenchyme around week 9. By week 14, the ICC forma network surrounding the MP (Fu et al. 2004; Wallace andBurns 2005). Whether ICC differentiation requires neuralcrest cells has not been clearly established, although somerecent studies have identified ICC in the absence of neuralcrest cells (Newman et al. 2003).

ICC have a central place in research aimed at examiningoesophageal contractions and the etiology and pathogenesis ofvarious motility disorders (Faussone-Pellegrini and Cortesini1985a; Roberts et al. 2008; Shafik et al. 2005; Khelif et al.2003). Histopathological studies on gastrointestinal stromaltumours have shown that they are immunopositive for thec-kit protein (Min and Leabu 2006), with the oesophagusbeing a rare site accounting for between 1% and 2% of allcases.

The object of the present study was to investigate thetiming of the appearance and distribution of ICC populationsin human embryonal and fetal oesophagi, in parallel with thedifferentiation of nerve structures and SMCs. The specimenswere exposed to Kit antibodies, whereas enteric plexuses wereimmunohistochemically examined by using anti-neuron-specific enolase (NSE). SMC differentiation was studiedimmunohistochemically with antibodies raised against α-smooth-muscle actin (αSMA) and desmin.

Materials and methods

Human material was obtained, according to the principlesof the Ethical Committee of the Faculty of Medicine of theUniversity of Nis, after legal abortions (0.5−1 h postmor-tem) and premature births attributable to pre-partial deaths.Both sexes were represented in the sample and no speci-mens had gastrointestinal disorders. Gestational ages wereestimated by anatomical criteria according to the CarnegieStaging system and the crown-rump length, head circum-ference and foot length. Each oesophageal specimen wasfixed in 10% neutral formalin for 24 h and paraffin-embedded. Routine histopathological examination of sec-tions stained with haematoxylin and eosin confirmed thatthe oesophageal wall morphology was normal in all cases.The study was approved by the Ethics Committee of theFaculty of Medicine of the University of Nis.

Study material consisted of seven human embryos and 23human fetuses at 7-27 weeks gestational age (7 weeks, n=3;8 weeks, n=4; 9 weeks, n=2; 10 weeks, n=3; 12 weeks, n=2,14 weeks, n=2; 15 weeks, n=3; 16-20 weeks, n=4; 21-24 weeks, n=4; 25-27 weeks, n=3). Embryos and smallfetuses (9 and 10 weeks) were processed completely,sequentially sectioned at a thickness of 4 μm and stained.

428 Cell Tissue Res (2010) 340:427–436

In older specimens between 12 and 27 weeks of gestation,the oesophagus was dissected free from the surroundingtissue and divided into the upper, middle and lower thirds.The sections were deparaffinised in xylol and a descendingseries of alcohol rinses (<1 min each) and then rehydrated indistilled water. Endogenous peroxidase was blocked with 3%H2O2 for 10 min at room temperature. This was followed byincubation with the primary antibodies for 60 min at roomtemperature and rinses in a phosphate-buffered solution(0.1 M PBS, pH 7.4). The primary antibodies were dissolvedin Dako antibody diluent (catalogue no. S0809; Dako NorthAmerica, Carpinteria, Calif., USA). The sections wereincubated with streptavidin-horseradish-peroxidase conju-gate for 30 min at room temperature. The complex wasvisualised with DAKO Liquid DAB + Substrate/ChromogenSystem (code no. K3468; Dako) and DAKO AEC +Substrate/Chromogen System (code no. K3469; Dako; seeFigs. 1g, h, 2c). All immunolabelled sections were counter-stained with Mayer’s haematoxylin. Immunoreactivity wasabsent in negative controls in which the primary antibodyhad been omitted. Sections were examined with an OlympusBX50 microscope and photographed with an Olympus PM-C35 camera.

The primary antibodies used and their respectivedilutions are listed in Table 1.

Results

At week 7 of development, numerous c-kit-immunoreactive(IR) cells were observed along the entire length of theoesophagus. These cells were present in the outer layers ofthe wall in the form of a wide belt of cells (Fig. 1a). Nodifferences were detected in the distribution of these cells inparticular oesophageal thirds. The c-kit-IR cells werepleomorphic with large round or oval nuclei. Numerouscellular processes extended from the body outwards andwere connected with each other to form a three-dimensionalnetwork (Fig. 1d). Within the belt of c-kit-IR cells, clearlydelineated groups of c-kit-negative cells were found that wereNSE-IR, confirming that they represented the primordia of theMP ganglia.

In developmental week 8, c-kit-IR cells form anuninterrupted sequence, a band of cells at the level of theMP (Fig. 1b). These cells envelop the ganglia, but neitherthe cells nor the cell processes are present within theganglia. The distribution is identical along the entireoesophagus. The c-kit-IR cells were pleomorphic, but incontrast to week 7, a large number of elongated spindle-shaped cells were present (Fig. 1b). These cells wererestricted to the areas surrounding the ganglia andcontained two long processes originating from the oppositeends.

By weeks 9-10, differences were observed in thedistribution of c-kit-IR cells in particular esophageal thirds.In the upper third, the c-kit-IR cells were scarce and presentat the level of the MP as individual spindle-shaped cells(Fig. 1e). In the middle and lower thirds of the oesophagus,the c-kit-IR cells were present around the myenteric gangliaand in the outer parts of the circular muscular layer(Fig. 1c). These cells were most commonly spindle-shaped with extended tapering processes in oppositedirections (Fig. 1f). The dendriform c-kit-IR cells withnumerous processes were much rarer.

At weeks 11-12, the differences in the distribution of c-kit-IR cells in the various esophageal thirds were morepronounced. In the lower third of the oesophagus, the c-kit-IR cells were present within the entire circular muscle layerand at the level of the MP. Within the circular layer, the c-kit-IR cells were spindle-shaped. The c-kit-IR cells wereparallel to the longitudinal axis of the adjacent muscle cellsand corresponded to the ICC-CM (Fig. 1h). The c-kit-IRcells were much rarer at the level of the MP compared withthe embryonic period of development. In the middle thirdof the oesophagus, the c-kit-IR cells were distributed in thesame way as in the lower third, although were fewer innumber, whereas in the upper third of the oesophagus, thec-kit-IR cells were rare. In addition to the ICC, we alsofound a large number of c-kit-IR mast cells but these wereeasily distinguished from the ICC on the basis of theirshape and granular content (Fig. 1g).

By 13-16 weeks, the differences in the distribution of thec-kit-IR cells in the three oesophageal regions were muchmore pronounced. The ICC-CM were abundant in the lowerthird of the oesophagus, less numerous in the middle thirdand rare in the upper third of the oesophagus. During thisperiod, the muscle cells of the circular layer were groupedinto muscle bundles and were enveloped in a thin septacomposed of connective tissue. The c-kit-IR cells werepresent inside the septa in the proximal, middle and distalthirds of the oesophagus. The c-kit-IR cells were mostlymultipolar and, rarely, bipolar cells with long processes.The c-kit-IR cells often lay perpendicular to the direction ofthe muscle bundles that they enveloped (Fig. 2a) andcorresponded to ICC-SEP. The c-kit-IR cells located withinthe poorly developed longitudinal muscle layer weredesignated as ICC-LM. These cells were spindle-shapedwith two extensions on their opposite ends and wereoriented parallel to the major axis of the muscle cells ofthe longitudinal layer.

In the period from week 17 to week 25 of develop-ment, differences were observed in the number anddistribution of ICC types in the oesophageal portions,which differed from each other on the basis of thecomposition of the muscularis propriae. These portionsdid not topographically match the corresponding oeso-

Cell Tissue Res (2010) 340:427–436 429

phageal thirds. Because of this, we use the followingterms in our subsequent descriptions: striated muscleportion, mixed muscle portion and smooth muscle potionof the oesophagus.

The striated muscle portion of the oesophagus wascomposed of striated muscle and was always shorter thanthe upper third. In this portion, the c-kit-IR ICC wereextremely rare and situated in the connective tissue between

Fig. 1 Immunohistochemistry for c-kit. a Lower third, 7 weeks. C-kit-immunoreactive (IR) cells (arrows) are located in the outer layersof the developing oesophagus (sm submucosa). b Middle third,8 weeks. C-kit-IR cells are restricted to the areas surrounding thepresumptive myenteric ganglia (mp myenteric plexus, l lumen). cMiddle third, 10 weeks. C-kit-IR cells are present around themyenteric ganglia and in the outer parts of the circular muscular layer(cm). d Lower third, 7 weeks. Pleomorphic c-kit-IR cells (arrows)with thin processes lying at the myenteric plexus level. e Upper third,

10 weeks. One spindle-shaped c-kit-IR cell (arrow) is located externalto the circular layer. f Lower third, 10 weeks. One spindle-shaped c-kit-IR cell with two processes (arrow) is closely apposed to amyenteric ganglion. g Lower third, 12 weeks. An isolated oval c-kit-IR mast cell is seen in the submucosa. h Lower third, 12 weeks.Interstitial cells of Cajal (ICC) of the circular muscle (ICC-CM) arebipolar and run parallel to the circular muscle cells. i Lower third,15 weeks. ICC-CM are numerous within the circular muscle layer. Bar100 µm (a, i), 50 µm (b, c, h), 20 µm (d–g)

430 Cell Tissue Res (2010) 340:427–436

Fig. 2 Immunohistochemistry for c-kit. a Middle third, 15 weeks.Two elongated ICC of the septa (ICC-SEP; arrows) lie perpendicularto the direction of the muscle bundles that they envelope (mb musclebundle, mp myenteric plexus). b Middle third, 16 weeks. Crosssection of the circular muscle layer with two ICC-CM (arrows) withinmuscle bundles. ICC-CM are oriented parallel to the long axis of themuscle cells. c Lower smooth part, 17 weeks (sm submucosa). ICC areabundant within the circular muscle layer (cm) and less numerouswithin the longitudinal muscle layer (lm). d Upper striated part,20 weeks. One ramified ICC-SEP (arrow) is located withinconnective-tissue septa. e Middle mixed part, 21 weeks. An elongated

ICC-SEP (arrow) with long processus lies perpendicular to thedirection of the muscle bundle that it envelopes. f Middle mixed part,21 weeks. One spindle-shaped ICC of the longitudinal muscle (ICC-LM; arrow) with two primary processes is located within thelongitudinal muscle layer. g Middle mixed part, 20 weeks. FourICC-CM (arrows) are interconnected and oriented parallel to the longaxis of the muscle cells. h Lower smooth part, 25 weeks. Intramus-cular ICC (ICC-IM) are present within the circular and longitudinalmuscle layers but no ICC-MP occur at the myenteric plexus level. Bar20 µm (a, b, d, f), 100 μm (c), 50 μm (e, h), 25 μm (g)

Cell Tissue Res (2010) 340:427–436 431

the muscle bundles (Fig. 2d). These cells corresponded tothe ICC-SEP. They were pleomorphic and showed anirregular appearance characterised by numerous processesextending in all directions (Fig. 2d). The ICC processescommonly branched further into secondary processes,which were also c-kit-IR. ICC were not present within themuscle bundles or around MP ganglia.

In the mixed muscle portion of the oesophagus, amixture of striated and smooth muscles was present. Thisportion was composed of the middle third and parts of theupper and lower third adjacent to it. ICC-CM, ICC-SEP andICC-LM were present in this portion. The ICC-CM laybetween the smooth and striated muscle cells and theirappearance was similar to that of the SMCs; they werespindle-shaped and appeared to be interconnected into longrows, extending parallel to the longitudinal axis of themuscle bundles (Fig. 2g). The ICC-SEP were mostlymultipolar and, rarely, bipolar cells with long processes(Fig. 2e); they showed an irregular appearance character-ised by numerous processes that extended in all directionsand were connected to each other to form a network aroundthe muscle bundles. The ICC-LM were rare in the middlethird of the oesophagus; they were often elongated, withtwo long processes that rarely branched, but resembled thecells described in the circular layer and in the distaloesophagus (Fig. 2f).

The smooth muscle portion of the oesophagus wascomposed of smooth muscle of various width and wasalways shorter than the lower third of the oesophagus. ICC-CM, ICC-SEP and ICC-LM were present in the smoothmuscle portion (Fig. 2c, g). The ICC-CM closely resembledthe above-described cells and were especially abundant inthe lower oesophageal sphincter (Fig. 2c). The ICC-SEPwere similar to the cells described in the mixed muscleportion. The ICC-LM were spindle-shaped and lay parallelto the direction of the muscle cells. The ICC-LM weresmaller than the ICC-LM present in the mixed portion. c-kit-IR ICC were not present around the myenteric ganglia(Fig. 2h).

NSE immunoreactivity

At week 7 of development, NSE immunoreactivity waspresent in smaller groups of cells situated in the externallayer of the oesophageal wall, representing the buds of the

MP ganglia (Fig. 3a). The distribution was identical in allportions of the oesophagus. At week 9 of development,NSE immunoreactivity was present in the MP region, but inthe groups of cells lying at the submucous border of thecircular layer and representing the buds of the submucousplexus ganglia. Both the myenteric and submucous plexus-es were present and intensely labelled in fetuses at 10 weeksof gestation (Fig. 3b). In older subjects, nerve structureswere intensely labelled and clearly visualised but theganglia were more numerous in the smooth muscle portionof the oesophagus than in the striated muscle portion of theoesophagus.

Desmin immunoreactivity and α-smooth muscle actinimmunoreactivity in muscle layers

At 7 weeks of gestation, desmin immunoreactivity was faintand present in the form of a thin row of cells that formed acircular layer (Fig. 3c). During this period, desminimmunoreactivity was not present outside the region ofthe MP at the position of the future longitudinal musclelayer. By 9 weeks of gestation, immunoreactivity fordesmin and α-SMA was present in the circular andlongitudinal muscular layers, although the latter was narrow(Fig. 3d, e). Immunoreactivity for desmin and α-SMA waspresent in the oesophagus in more advanced ages and anydifferences in development between the circular andlongitudinal muscular layer were diminished (Fig. 3f).

Discussion

In the present study, we have determined the pattern ofdifferentiation and distribution of c-kit-IR ICC in embry-onic and fetal oesophagi by using immunohistochemistry.The results of the study demonstrate that c-kit-IR cells arepresent in the oesophagus by week 7 of development at thelevel of the MP. In view of their number and distributionthroughout the oesophagus, the possibility of the presenceof these cells even before this period cannot be excluded,although we do not have any data to support this. At week7 of gestation, c-kit-IR cells are also present in the stomachwall, around the MP elements, whereas c-kit-IR cellsappear later in the small and large bowel (in the smallbowel at 9 weeks and in the colon at 10-12 weeks; Kenny

Antigen Clone Supplier Dilution

C-kit CD-117 Dako 1:300

α-SMA (α-smooth-muscle actin) 1-A4 Dako 1:100

NSE (neuron-specific enolase) BBS/NS VI-H14 Dako 1:100

Desmin DE-R-11 Dako 1:100

Table 1 Antibodies

432 Cell Tissue Res (2010) 340:427–436

et al. 1999; Wester et al. 1999; Wallace and Burns 2005;Radenkovic et al. 2009). This finding corroborates thehypothesis that c-kit-IR cells appear in the digestive tractorgans following a rostrocaudal gradient, in a way similarto the MP elements (Fu et al. 2004; Wallace and Burns2005).

The results obtained so far have shown that, in thedigestive tract as a whole, the MP appears first and, onlyafter some time, are c-kit-IR cells detectable in theimmediate vicinity (Fu et al. 2004; Radenkovic et al.2009; Faussone-Pellegrini et al. 2007). A similar develop-mental lag for the ICC has been reported in mouse embryos

Fig. 3 Immunohistochemistry for neuron specific enolase (a, b),desmin (c, d) and α-smooth-muscle actin (e, f). a Middle third,8 weeks (l lumen). Note the immunopositive myenteric plexuselements (arrows). b Lower third, 10 weeks (cm circular musclelayer, sm submucosa). Both the myenteric plexus (mp) and submucousplexus (arrow) are immunopositive. c Lower third, 8 weeks. Desminimmunoreactivity is present in the form of a belt of cells (arrows),

which incorporates the cells that will form the circular muscle layer. dMiddle third, 10 weeks. Both the circular (cm) and longitudinal(arrow) muscle layers are desmin-IR. e Lower third, 9 weeks. Thelongitudinal muscle layer is thin (arrow). f Middle mixed part,21 weeks. The longitudinal (lm) and circular muscle (cm) layers are ofalmost equal thickness. Bar 50 μm (a, b, d, e), 100 μm (c, f)

Cell Tissue Res (2010) 340:427–436 433

(Wu et al. 2000). As ICC are of mesenchymal origin, thesefindings indicate the possibility that an MP element iscrucial for ICC differentiation, probably as a source of SCF,which has been shown to be important for ICC differenti-ation (Ward et al. 1994). The ligand of the c-kit receptor(steel factor) is found in nerves (Torihashi et al. 1996;Young et al. 1998) and within the circular muscle layer(Lecoin et al. 1996). Furthermore, in the oesophagus andstomach cardia, c-kit-IR cells first appear in the MP region(Radenkovic et al. 2009), around the ganglia, and, in laterdevelopmental stages, the number is markedly reduced orcompletely disappears in the region. Whether ICC differ-entiation requires neural crest cells has not been clearlyestablished, although some recent studies have identifiedICC in the absence of neural crest cells (Wu et al. 2000;Newman et al. 2003).

Based on the results obtained during week 7 ofdevelopment, MP elements are present in the entireoesophagus, whereas the submucous plexus appears atweek 9. This finding is consistent with earlier studiesdemonstrating the development of the MP approximately2 weeks before the submucous plexus (Fu et al. 2004;Wallace and Burns 2005).

In agreement with previous studies (Wallace and Burns2005), the current study has shown that the circular musclelayer is the first to differentiate, followed by the longitu-dinal muscle layer, although some authors have claimedthat the muscle layers develop simultaneously (Fu et al.2004). The circular muscle layer becomes markedly wideruntil the 4th month of development, after which thedifference gradually diminishes.

The c-kit-IR cells that are present in the oesophagus atweek 7 of development differ markedly in shape from theICC described in samples of more advanced gestationalages (i.e. after week 10) and also from the cells described inthe oesophagus after birth and during the adult period. Asimilar observation has been reported in the stomach wall(Kenny et al. 1999; Radenkovic et al. 2009). Ourhypothesis is that the cells described at weeks 7 and8 belong to committed ICC progenitors (Huizinga andWhite 2008) and that the cells described later are c-kit-IRICC. One conclusion that can perhaps be drawn from all of

these findings is that these progenitors differentiate intoICC from weeks 8-10 of development. This hypothesis,however, requires immunohistochemical confirmation.

At weeks 7-8, the c-kit-IR cells are distributed along theentire oesophagus. The differences in distribution andnumber of ICC, in particular in the oesophageal portions,start to appear at the beginning of the 3rd month ofdevelopment. Starting from the 3rd month, the number ofthese cells is significantly higher in the lower thirdcompared with the middle third, with the number beingthe lowest in the upper third of the oesophagus. During the4th and 5th months, the relationship is established thatexists in later stages of development (Table 2).

The ICC are also distributed in a similar way during theadult period (Torihashi et al. 1999) and in the catoesophagus (Huizinga et al. 2008). From the 5th monthonwards, i.e. during the later stages of development, c-kitIR ICC are extremely rare or completely missing around themyenteric ganglia, i.e. ICC-MY are absent throughoutthe oesophagus, although the c-kit-IR cells first appear atthe level of the MP.

In the smooth muscle portion of the oesophagus, ICC-CM are particularly numerous, especially in the terminalpart corresponding to the lower oesophageal sphincter,which contains a wide circular muscle layer. This finding,together with the absence of the ICC-MY, is almostidentical to the ICC distribution in the stomach cardia(Radenkovic et al. 2009). Numerous ICC-CM have beendescribed as being similarly distributed in the pyloricsphincter region (Ward et al. 1998; Piotrowska et al.2003). Previous data suggest that the intramuscular ICC(ICC-IM) play an important role in NO-dependent neuro-transmission in the lower oesophageal sphincter and pyloricsphincter. Loss of the ICC-IM may interfere with therelaxation and normal motility in these sphincters (Ward etal. 1998; Farre et al. 2007).

ICC-CM, ICC-SEP and ICC-LM are present in the fetaloesophagus but ICC-MY are absent. These cell types havebeen described in adult human and cat oesophagi, makingthe latter a suitable model for the human oesophagus. Inview of the function of ICC-IM and ICC-SEP in other partsof the digestive tract, our findings support the existing

Table 2 Distribution of interstitial cells of Cajal (ICC) immunoreactive ofr c-kit in fetal oesophagus during the 5th month of development (CMcircular muscle, LM longitudinal muscle, SEP septa, + present, ++ numerous, +++ highly numerous, ND not detected)

Cell types Striated muscle portion Mixed muscle portion Smooth muscle portion

CM LM CM LM CM LM

ICC-CM ND ND ++ ND +++ ND

ICC-SEP + ND + ND ++ ND

ICC-LM ND ND ND + ND ++

434 Cell Tissue Res (2010) 340:427–436

hypothesis that, in the oesophagus, ICC might act asmechanosensors (Thuneberg and Peters 2001; Rumessenet al. 2001; Fox et al. 2002), representing specialisedspindle proprioceptors and might participate in signaltransmission (Ward et al. 1998; Wang et al. 2003). Apacemaking role for the oesophageal ICC can probably beruled out.

All of the described oesophageal c-kit-IR ICC can becategorised into the following two morphological types: (1)bipolar cells that extend tapering processes in oppositedirections and (2) positive cells that are mostly multipolarcells, being irregularly shaped and with long processes thatcommonly branch further into secondary processes. AllICC-IM (ICC-CM and ICC-LM) belong to the firstmorphological type. This applies both to the cells withinSMCs and to those within the bundles composed of striatedmuscles. The ICC-SEP most commonly belong to thesecond morphological type.

In conclusion, c-kit-IR cells appear in the humanoesophagus in the MP region, with the number diminishingin the region later on; they occur within the muscle layersand connective septa surrounding the muscle bundles. Bythe 5th month of development, the ICC are distributedthroughout the oesophagus in a way similar to that of theadult oesophagus.

Acknowledgements We thank Mile Randjelovic for his technicalassistance. We also thank Milkica Tosic Djordjevic for her expert helpwith the immunohistochemical procedures.

References

Beckett EAH, McGeough CA, Sanders KM, Ward SM (2003) Pacingof interstitial cells of Cajal in the murine gastric antrum: neurallymediated and direct stimulation. J Physiol (Lond) 553:545–559

Beckett EAH, Takeda Y, Yanase H, Sanders KM, Ward SM (2005)Synaptic specializations exist between enteric motor nerves andinterstitial cells of Cajal in the murine stomach. J Comp Neurol493:193–206

Belzer V, Kobilo T, Rich A, Hanani M (2002) Intercellular couplingamong interstitial cells of Cajal in the guinea pig small intestine.Cell Tissue Res 307:15–21

Blank EL, Greenwood B, Dodds WJ (1989) Cholinergic control ofsmooth muscle peristalsis in the cat esophagus. Am J PhysiolGastrointest Liver Physiol 257:G517–G523

Burns AJ, Lomax AEJ, Torihashi S, Sanders KM, Ward SM (1996)Interstitial cells of Cajal mediate inhibitory neurotransmission inthe stomach. Proc Nat Acad Sci USA 93:12008–12013

Burns AJ, Herbert TM, Ward SM, Sanders KM (1997) Interstitial cellsof Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry. Cell Tissue Res 290:11–20

Diamant NE (1997) Neuromuscular mechanisms of primary peristal-sis. Am J Med 103:40S–43S

Farre R, Wang XY, Vidal E, Domenech A, Pumarola M, Clave P,Huizinga JD, Jimenez M (2007) Interstitial cells of Cajal andneuromuscular transmission in the rat lower oesophageal sphinc-ter. Neurogastroenterol Motil 19:484–496

Faussone-Pellegrini MS, Cortesini C (1985a) The muscle coat of thelower esophageal sphincter in patients with achalasia andhypertensive sphincter. An electron microscopic study. J Sub-microsc Cytol 17:673–685

Faussone-Pellegrini MS, Cortesini C (1985b) Ultrastructural featuresand localization of the interstitial cells of Cajal in the smoothmuscle coat of human esophagus. J Submicrosc Cytol 17:187–197

Faussone-Pellegrini MS, Vannucchi MG, Alaggio R, Strojna A,Midrio P (2007) Morphology of the interstitial cells of Cajal ofthe human ileum from foetal to neonatal life. J Cell Mol Med11:482–494

Fox EA, Phillips RJ, Byerly MS, Baronowsky EA, Chi MM, PowleyTL (2002) Selective loss of vagal intramuscular mechanorecep-tors in mice mutant for steel factor, the c-Kit receptor ligand.Anat Embryol 205:325–342

Fu M, Tam PK, Sham MH, Lui VC (2004) Embryonic development ofthe ganglion plexuses and the concentric layer structure of humangut: a topographical study. Anat Embryol 208:33–41

Hagger R, Gharaie S, Finlayson C, Kumar D (1998) Regional andtransmural density of interstitial cells of Cajal in human colonand rectum. Am J Physiol 275:G1309–G1316

Hanani M, Farrugia G, Komuro T (2005) Intercellular coupling ofinterstitial cells of Cajal in the digestive tract. Int Rev Cytol242:249–282

Huizinga JD, White EJ (2008) Progenitor cells of interstitial cells ofCajal: on the road to tissue repair. Gastroenterology 134:1252–1254

Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB,Bernstein A (1995) W/kit gene required for interstitial cells ofCajal and for intestinal pacemaker activity. Nature 373:347–349

Huizinga JD, Reed DE, Berezin I, Wang XY, Valdez DT, Liu LWC,Diamant N (2008) Survival dependency of intramuscular ICC onvagal afferent nerves in the cat esophagus. Am J Physiol RegulIntegr Comp Physiol 294:R302–R310

Kallmunzer B, Sorensen B, Neuhuber WL, Worl J (2008) Enteric co-innervation of striated muscle fibers in human oesophagus.Neurogastroenterol Motil 20:597–610

Kenny SE, Connell G, Woodward MN, Lloyd DA, Gosden CM,Edgar DH, Vaillant C (1999) Ontogeny of interstitial cells ofCajal in the human intestine. J Pediatr Surg 34:1241–1247

Khelif K, De Laet MH, Chaouachi B, Segers V, Vanderwinden JM(2003) Achalasia of the cardia in Allgrove’s (triple A) syndrome:histopathologic study of 10 cases. Am J Surg Pathol 27:667–672

Komuro T (2006) Structure and organization of interstitial cells ofCajal in the gastrointestinal tract. J Physiol (Lond) 576:653–658

Kuo B, Urma D (2006) Esophagus—anatomy and development. GIMotility online. doi:10.1038/gimo6

Lecoin L, Gabella G, Le Douarin N (1996) Origin of the c-kit-positiveinterstitial cells in the avian bowel. Development 122:725–733

Maeda H, Yamagata A, Nishikawa S, Yoshinaga K, Kobayashi S,Nishi K (1992) Requirement of c-kit for development ofintestinal pacemaker system. Development 116:369–375

Min KW, Leabu M (2006) Interstitial cells of Cajal (ICC) andgastrointestinal stromal tumor (GIST): facts, speculations, andmyths. J Cell Mol Med 10:995–1013

Mitsui R, Komuro T (2002) Direct and indirect innervation of smoothmuscle cells of rat stomach, with special reference to theinterstitial cells of Cajal. Cell Tissue Res 309:219–227

Newman CJ, Laurini RN, Lesbros Y, Reinberg O, Meyrat BJ (2003)Interstitial cells of Cajal are normally distributed in bothganglionated and aganglionic bowel in Hirschsprung’s disease.Pediatr Surg Int 19:662–668

Piotrowska AP, Solari V, Puri P (2003) Distribution of hemeoxygenase-2 in nerves and interstitial cells of Cajal in the normalpylorus and in infantile hypertrophic pyloric stenosis. ArchPathol Lab Med 127:1182–1186

Cell Tissue Res (2010) 340:427–436 435

Preiksaitis HG, Diamant NE (1999) Myogenic mechanism forperistalsis in the cat esophagus. Am J Physiol Gastrointest LiverPhysiol 277:G306–G313

Radenkovic G, Savic V, Mitic D, Grahovac S, Bjelakovic M, Krstic M(2009) Development of c-kit immunopositive interstitial cells ofCajal in the human stomach. J Cell Mol Med. doi:10.1111/j.1582-4934.2009.00725.x

Roberts CGP, Hummers LK, Ravich WJ, Wigley FM, Hutchins GM(2008) A case–control study of the pathology of oesophagealdisease in systemic sclerosis (scleroderma). Gut 55:1697–1703

Rumessen JJ, Kerchove d’Exaerde A de, Mignon S, Bernex F,Timmermans J-P, Schiffmann SN, Panthier JJ, Vanderwinden JM(2001) Interstitial cells of Cajal in the striated musculature of themouse esophagus. Cell Tissue Res 306:1–14

Shafik A, Ahmed I, Sibai OE, Shafik AA (2005) Interstitial cells ofCajal in reflux esophagitis: role in the pathogenesis of thedisease. Med Sci Monit 11:452–456

Suzuki H, Ward SM, Bayguinov YR, Edwards FR, Hirst GDS (2003)Involvement of intramuscular interstitial cells in nitrergicinhibition in the mouse gastric antrum. J Physiol (Lond)546:751–763

Thuneberg L, Peters S (2001) Toward a concept of stretch-coupling insmooth muscle. I. Anatomy of intestinal segmentation and sleevecontraction. Anat Rec 262:110–124

Torihashi S, Ward SM, Nishikawa S-I, Nishi K, Kobayashi S, SandersKM (1995) C-kit-dependent development of interstitial cells andelectrical activity in the murine gastrointestinal tract. Cell TissueRes 280:97–111

Torihashi S, Yoshida H, Nishikawa S, Kunisada T, Sanders KM(1996) Enteric neurons express Steel factor-lacZ transgene in themurine gastrointestinal tract. Brain Res 738:323–328

Torihashi S, Horisawa M, Watanabe Y (1999) c-Kit immunoreactiveinterstitial cells in the human gastrointestinal tract. J Auton NervSyst 75:38–50

Wallace AS, Burns AJ (2005) Development of the enteric nervoussystem, smooth muscle and interstitial cells of Cajal in the humangastrointestinal tract. Cell Tissue Res 319:367–382

Wang XY, Paterson C, Huizinga JD (2003) Cholinergic and nitrergicinnervation of ICC-DMP and ICC-IM in the human smallintestine. Neurogastroenterol Motil 15:531–543

Ward SM, Sanders KM (2001) Physiology and pathophysiology of theinterstitial cells of Cajal: from bench to bedside. Am J PhysiolGastrointest Liver Physiol 281:G602–G611

Ward SM, Burns AJ, Torihashi T, Sanders KM (1994) Mutation of theproto-onkogene c-kit blocks development of interstitial cells andelectrical rhythmicity in murine intestine. J Physiol (Lond)480:91–97

Ward SM, Morris G, Reese L, Wang XY, Sanders KM (1998)Interstitial cells of Cajal mediate enteric inhibitory neurotrans-mission in the lower esophageal and pyloric sphincters. Gastro-enterology 115:314–329

Wester T, Eriksson L, Olsson Y, Olsen L (1999) Interstitial cells ofCajal in the human fetal small bowel as shown by c-kitimmunohistochemistry. Gut 44:65–71

Williams DE, Eisenmann J, Baird A (1990) Identification of a ligandfor the c-kit proto-oncogene. Cell 63:167–174

Wu JJ, Rothman TP, Gershon MD (2000) Development of theinterstitial cell of Cajal: origin, kit dependence and neuronaland nonneuronal sources of kit ligand. J Neurosci Res 59:384–401

Wu M, Majewski M, Wojtkiewicz J, Vanderwinden JM, AdriaensenD, Timmermans JP (2003) Anatomical and neurochemicalfeatures of the extrinsic and intrinsic innervation of the striatedmuscle in the porcine esophagus: evidence for regional andspecies differences. Cell Tissue Res 311:289–297

Young HM, Torihashi S, Ciampoli D, Sanders KM (1998) Identifica-tion of neurons that express stem cell factor in the mouseintestine. Gastroenterology 115:898–908

436 Cell Tissue Res (2010) 340:427–436