Esophageal development and epithelial homeostasis...Esophageal development and epithelial homeostasis Sanne L. Rosekrans, Bart Baan, Vanesa Muncan, and Gijs R. van den Brink Tytgat

Esophageal development and epithelial homeostasis

Sanne L. Rosekrans, Bart Baan, Vanesa Muncan, and Gijs R. van den BrinkTytgat Institute for Liver and Intestinal Research and Department of Gastroenterology and Hepatology, Academic MedicalCenter, Amsterdam, the Netherlands

Submitted 18 March 2015; accepted in final form 25 June 2015

Rosekrans SL, Baan B, Muncan V, van den Brink GR. Esophageal devel-opment and epithelial homeostasis. Am J Physiol Gastrointest Liver Physiol 309:G216–G228, 2015. First published July 2, 2015; doi:10.1152/ajpgi.00088.2015.—The esophagus is a relatively simple organ that evolved to transport food andliquids through the thoracic cavity. It is the only part of the gastrointestinal tract thatlacks any metabolic, digestive, or absorptive function. The mucosa of the adultesophagus is covered by a multilayered squamous epithelium with a remarkablesimilarity to the epithelium of the skin despite the fact that these tissuesoriginate from two different germ layers. Here we review the developmentalpathways involved in the establishment of the esophagus and the way thesepathways regulate gut-airway separation. We summarize current knowledge ofthe mechanisms that maintain homeostasis in esophageal epithelial renewal inthe adult and the molecular mechanism of the development of Barrett’smetaplasia, the precursor lesion to esophageal adenocarcinoma. Finally, weexamine the ongoing debate on the hierarchy of esophageal epithelial precursorcells and on the presence or absence of a specific esophageal stem cellpopulation. Together the recent insights into esophageal development andhomeostasis suggest that the pathways that establish the esophagus duringdevelopment also play a role in the maintenance of the adult epithelium. We arebeginning to understand how reflux of gastric content and the resulting chronicinflammation can transform the squamous esophageal epithelium to columnarintestinal type metaplasia in Barrett’s esophagus.

esophagus; development; homeostasis; stem cell; endoderm

THE WORD ESOPHAGUS IS DERIVED from the Greek words ��ε��(oisein, to carry) and ��ε�� (phagein, to eat). This descriptionfits well with the functional role of the esophagus that mainlyserves to “carry food” into the stomach. From the pharyn-goesophageal junction, the esophagus passes through the me-diastinum and diaphragm and connects to the cardia of thestomach at the gastroesophageal junction or Z-line. Thepharyngoesophageal and gastroesophageal junctions anatomi-cally overlap with the upper and lower esophageal sphincters.Both sphincters are closed except during swallowing to assurea unidirectional flow of esophageal content toward the stomachand to prevent reflux of gastric content into the esophagus. Therelatively simple histology of the esophageal epithelium cor-responds with the fact that the esophagus has no role other thanto pass food through the thorax to the stomach. It does not playa known digestive, endocrine, or metabolic role and the epi-thelium consists of a simple stratified squamous epithelium,which provides a good protective layer against the unmodifiedfood stream on its way to the stomach. Despite the perhapssomewhat prosaic functional role of the esophagus comparedwith other organs in the body, we feel that it is essential to gaina better understanding of the mechanisms that regulate normal

esophageal homeostasis. Esophageal cancer is a disease with adismal prognosis given that the incidence rate in the USA is4.6/100,000 whereas the mortality rate is 4.4/100,000, indicat-ing a mortality of around 95% for the disease. A betterunderstanding of the pathways that maintain esophageal epi-thelial homeostasis and the way these pathways are deregulatedduring oncogenesis may provide novel approaches to treatmentof esophageal cancer. In this review we aim to give anoverview of the current understanding of the mechanismsinvolved in esophageal development and homeostasis.

DEVELOPMENT OF THE ESOPHAGUS

Normal Esophageal Morphogenesis and EndodermalDifferentiation

The esophagus develops from the foregut. A critical phase ofesophageal morphogenesis is when the respiratory appendagestarts to form from the foregut tube at mouse embryonic day(E)9.5 and human E26 (Fig. 1). The respiratory appendageconsists of a central ventral tracheal bud and two adjacentventrolateral lung buds (12, 57, 82). The airways will separatefrom the esophagus by a process of elongation and septation, aprocess that is completed by mouse E11.5. The esophagus andtrachea show distinct endodermal and mesenchymal develop-ment. The trachea will develop pseudostratified columnar ep-ithelium and is enveloped in cartilage rings ventrally. Theesophagus will form multilayered squamous epithelium and theesophageal mesenchyme develops the smooth muscle layer

Address for reprint requests and other correspondence: G. R. van den Brink,Dept. of Gastroenterology & Hepatology, Rm. C2-115, Academic MedicalCenter, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands (e-mail:[email protected]).

Am J Physiol Gastrointest Liver Physiol 309: G216–G228, 2015.First published July 2, 2015; doi:10.1152/ajpgi.00088.2015.Review

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required for esophageal motility and propulsion of food. Theexact mechanism of foregut separation into esophagus andtrachea has not been examined by in vivo imaging and remainsa matter of debate (20).

When the mouse esophagus has been clearly established byE11.5, the epithelium consists of a single keratin (K)8-positivecuboidal epithelial layer (Fig. 2) (100). In the following days(E13.5–E17.5) the epithelium gradually becomes more layereduntil there are around four layers of epithelial cells (71, 100).During development there is a gradual conversion of a K8-

positive cuboidal epithelium to a K14-positive squamous epi-thelium. This process begins at the basal layer around dayE17.5 and the basal layer is mostly K14 positive at birth (100).The suprabasal layers will subsequently gradually lose K8expression postnatally (100). The onset of squamous celldifferentiation can also be observed at the suprabasal layers,which start to express the squamous cell differentiation markerinvolucrin around E15.5. Expression of the late differentiationmarker K10 starts at postnatal day (P)1 (100). During devel-opment the epithelium contains numerous ciliated cells, which

Fig. 1. Esophageal and airway development from the endoderm. A–C: the esophagus will develop from the dorsal part and the respiratory tract from thecounterpart localized at the ventral side. D–H: common types of esophageal atresia and/or tracheoesophageal fistula. D: esophageal atresia. E: tracheoesophagealfistula. F: esophageal atresia with distal tracheoesophageal fistula. G: esophageal atresia with proximal tracheoesophageal fistula. H: esophageal atresia withdouble tracheoesophageal fistula.

Fig. 2. Development of the esophageal epithelium. By embryonic day (E)11.5 the epithelium consists of a single cuboidal epithelial layer. All cells are positivefor K8 (depicted in pink). Around E17.5 cells start to lose expression of K8 (orange reflects K8-negative cells). Gradually basal cells start to express K14 (purple).Based on the model described in Yu et al. (100).

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have almost completely disappeared by P4 (52, 71). In theadult mouse the epithelium will undergo a process of kerati-nization; this does not occur in humans.

Recently, Wang et al. (93) proposed a model in which theembryonic cuboidal epithelium is displaced by an underminingpopulation of p63-positive squamous cell precursors that mi-grates under the cuboidal epithelial cells from the proximal todistal esophagus. Wang et al. propose that the squamous cellprogenitors thus outcompete cuboidal progenitors by displac-ing them from access to the basement membrane. This is aninteresting hypothesis but so far the evidence is circumstantial.Wang et al. used terms such as tracing and tracking for theirstudy of the behavior of the squamous vs. cuboidal cells. Itshould be stressed that no actual lineage tracing was performedof either population. In lineage tracing experiments a definedpopulation of cells is genetically irreversibly marked so that thefate of the cells can be traced irrespective of changes in thephenotype. In the experiments of Wang et al. squamous cellsand cuboidal cells were examined with cell lineage markers byimmunofluorescence at different time points of developmentonly. Such experiments do not demonstrate that a cell thatexpresses a cuboidal cell marker at one point in developmentwas not at the basal layer expressing a squamous cell markerthe day before. The experiments therefore by no means ex-cluded a scenario that cuboidal cells actually transdifferentiateto a squamous cell fate in a proximal-to-distal wave. Bycomparison, such a wave of differentiation is known to trans-form the intestinal epithelium from a cuboidal to a columnarepithelium along the proximodistal axis (87). Actual lineagetracing experiments are thus required to further examine theinteresting hypothesis by Wang et al.

Signaling Pathways Involved in Esophageal Morphogenesis

Patterning of cellular fate during development is dependenton positional information that couples the position of a cell toits function. Spatial information is laid down in a tissue by theformation of gradients of extracellular signals or so-calledmorphogens. Receptive cells will respond to the morphogen ina concentration-dependent manner, resulting in the expressionand activation of different transcription factors. The combinedactivity of these transcriptional regulators is one of the mostimportant determinants of cellular phenotype. Thus a cellularfunction will depend on a cells position in the concentrationgradient. In each tissue multiple gradients exist of differentmorphogens and their antagonists, allowing formation of intri-cately patterned tissues.

A limited number of morphogenetic signaling families areused in different constellations throughout development;this has been aptly termed the morphogenetic code (26).Four families of morphogenetic pathways can roughly bedistinguished: Wnt, Hedgehog (Hh), Tgf- families, and alarge group of receptor tyrosine kinases such as fibroblastgrowth factor, platelet-derived growth factor, and epidermalgrowth factor, which share similar intracellular signalingpathways. The incredible level of variation in tissue pattern-ing stems from the sheer infinite variation in which thesepathways are modified by gradients of various agonists andantagonists, differences in the expression of intracellulardownstream regulators of signaling output and, for example,differences in autocrine vs. paracrine signaling. For in-

stance, the Hedgehog signaling pathway is a mitogenicpathway that is involved in oncogenesis of the skin andbrain, where it acts in an autocrine fashion on the affectedcells. Similarly, in the adult esophagus a Hedgehog ligand isexpressed in the basal layer and signaling acts in a autocrinefashion on basal cells and stimulates their proliferation (seealso below in the Regulators of Esophageal EpithelialProliferation and Differentiation section) (89). In contrast,in the intestine signaling is uniquely from the differentiatedepithelial cells to the underlying mesenchyme. Here Hedge-hog signaling regulates survival and expansion of the mes-enchyme and in fact negatively regulates epithelial precur-sor cell proliferation (11).

As will be seen below, these morphogenetic pathways arecritical regulators of esophageal development. The functionof the different regulators of esophageal development iden-tified to date has been revealed by foregut abnormalitiesobserved in mouse mutants and in humans with congenitalabnormalities. Three different important gross structuralabnormalities can be observed in the various mutants (Fig.1). One is the improper separation of the esophagus andtrachea, leading to the development of tracheoesophagealfistula (TEF). Another is defective outgrowth of the airwaysresulting in a hypoplastic respiratory system. Finally, somemutants fail to maintain the esophageal tube, resulting inhypoplasia or atresia of the esophagus (esophageal atresia;see Table 1 for an overview of the mouse mutants discussedbelow).

Sox2 and Nkx2.1 Are Tissue Specific TranscriptionalRegulators of Gut-Airway Separation

Two tissue-specific transcriptional regulators have beenidentified that are specific markers of esophageal vs. airwayendoderm. Sox2 marks the endodermal cells that will form theesophagus and is expressed throughout the esophageal epithe-lium in the adult (69). Nkx2.1 identifies the endodermal cellsfrom which the respiratory tract will form and is expressed inalveolar epithelial cells in the adult (36). These transcriptionfactors not only are useful markers to identify esophageal vs.respiratory differentiation but also play a key role in theestablishment of the respective organs.

Nkx2.1 expression marks a population of cells in the anteriorforegut at E9.0 just before the formation of the respiratoryprimordium (36, 56). Hereafter Nkx2.1 will be expressed onlyin the respiratory primordium and in the developing airwaysand is excluded from the endoderm of the dorsal foregut tube.This dorsal foregut region is now marked by exclusive expres-sion of Sox2 and will develop into the esophagus (69). Micethat lack Nkx2.1 have an undivided foregut tube that connectsthe pharynx to the stomach (56). The most proximal part of thisNkx2.1 knockout foregut is enclosed by a few poorly devel-oped cartilage rings but the remainder of the mesenchyme ischaracterized by smooth muscle development as typical for theesophagus. In accordance, the endoderm expresses markers ofesophageal differentiation such as Sox2 and p63 (69). The lungbuds form but fail to undergo branching morphogenesis andform cystic structures that fail to express markers of lungdifferentiation (56). Thus Nkx2.1 marks the endodermal cellsthat will form the respiratory primordium. Furthermore,Nkx2.1 is required for the proper elongation and separation of

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the trachea, for branching organogenesis, and for differentia-tion of lung endoderm.

Sox2 marks the prospective esophageal foregut cells. As analternative for Sox2/ mice, which are embryonic lethal at theblastocyst stage (2), Que et al. (69) used a hypomorphicSox2-mutant mice to study the role of Sox2 in esophagealdevelopment. They reported that a large proportion of Sox2hypomorphic mice display fusion of the esophageal lumen withthe tracheal lumen (tracheoesophageal fistula) combined withloss of the proximal esophagus (atresia). In Sox2 hypomorphicanimals with an intact esophagus the esophageal diameter wasdiminished. At E18.5 the remaining esophagus was coveredwith columnar epithelium and lacked expression of esophagealmaturation markers, such as p63 and keratin 14. In contrast, theepithelium is strongly positive for Nkx2.1 and expresses mark-ers of airway differentiation. The importance of Sox2 inesophageal development is underscored by the fact that Sox2mutations cause tracheoesophageal fistula and esophageal atre-sia in humans (9, 95). This phenotype indicates that Sox2 isrequired to maintain the esophageal endoderm during devel-opment and represses Nkx2.1-mediated airway-type matura-tion.

Thus Sox2 and Nkx2.1 are critical regulators of esopha-geal vs. airway specification that are expressed in nonover-lapping patterns and repress each other’s expression andactivity.

p63 Is a Critical Regulator of Esophageal SquamousEpithelial Differentiation

The p53 homologue p63 is a critical transcriptional regulatorof squamous epithelial cell fate (13, 99). p63 plays a criticalrole in maintaining homeostatic proliferation of basal cells asp63 expression specifically marks the basal layer of squamoustissues (98) and p63/ mice completely lack stratified squa-mous epithelial tissues at birth. The esophagus in p63-mutantmice has a pseudostratified columnar epithelium that showssigns of respiratory maturation, such as presence of ciliated andgoblet cells (14, 99). Since the columnar epithelium observedin Sox2 hypomorphic mice was devoid of p63 expression, thismost likely indicates that Sox2 functions upstream of p63 in

the induction of a squamous phenotype in the esophagealendoderm.

The Morphogenetic Signaling Network Responsible for Gut-Airway Separation

As mentioned above, a limited number of morphogeneticsignaling pathways is used in patterning the tissues of ourbody. The major known morphogenetic pathways are all in-volved in endodermal-mesenchymal interactions during esoph-ageal development. As we will review below, there is anoticeable difference in the role of the various signaling path-ways between the developing esophagus and airways, andmany signaling defects in the pathways discussed below leadeither to a preferential esophageal or a predominant airwayphenotype. For clarity we will discuss the pathway separatelybut try to indicate interactions between the different pathwayswhere these are known. We will focus on phenotypes in mousemodels because these are often more insightful than mutationsin humans, in whom the many important mutations are missedbecause of embryonic lethality, and patients are rare anddifficult to characterize at the molecular level because of theinability to obtain tissue. For an excellent overview of thehuman mutations that result in foregut phenotypes we refer torecent reviews (8, 10).

Sonic Hedgehog signaling. The first morphogen identified asa critical regulator of gut-airway separation was Sonic Hedge-hog. Sonic Hedgehog (Shh) is initially expressed throughoutthe anterior endoderm but is restricted to the distal esophagusat later stages (42, 70). Gli transcription factors, which mediateHedgehog signaling, are selectively expressed in the meso-derm, indicating that Hedgehog signals exclusively in a para-crine manner from endoderm to mesoderm (28). The criticalrole of Shh signaling in foregut development was revealed inShh-mutant mice. The phenotype of Shh/ mice is remark-ably similar to Sox2-mutant mice. At E17.5 the proximalShh/ esophagus is hypoplastic and the developing tracheaand lungs fail to separate correctly from the gut. More distally(where Shh expression is highest in normal mice), there is nodiscernible remaining esophagus in Shh/ mice at this pointin development (42, 65). This suggests that paracrine Shh

Table 1. Mouse mutants

Mouse Model Foregut Phenotype Reference

Nkx2.1/ undivided foregut tube connecting the pharynx to the stomach, impaired lung bud development 56Sox2EGFP/COND majority develops TEF/EA 69, 95p63/ esophagus with pseudostratified columnar epithelium that shows signs of respiratory maturation 14, 99Shh/ hypoplastic proximal esophagus and septation defects 42, 65Gli2/ small esophageal lumen with poorly developed mesenchyme 58Gli2//Gli3�/ hypoplastic foregut and absent trachea and lung appendages 58Foxf1�/ TEF, narrow esophagus, and lung hypoplasia 46Noggin/ TEF/EA, with intact airway differentiation 41, 68Bmp4/ single tube connecting the pharynx to the stomach and hypoplastic lungs 40Bmp4�/ Noggin/ no TEF/EA was seen 41, 68Bmp7/ Noggin/ no TEF/EA was seen 41, 68Fgf10/ lung agenesis 16Fgfr2 IIIb/

Wnt2/2b/ Ctnnb1/ lung agenesis, with complete loss of trachea and lungs 24Wnt7blacZ/

lung hypoplasia 77Barx1/ septation defects 97Rar�/�2�/ septation defects and hypoplastic lungs 53

TEF, tracheoesophageal fistula; EA, esophageal atresia.

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signaling to the mesenchyme is required to allow the properelongation and survival of esophageal tissue and to maintain amesenchymal barrier between the developing tubes of theesophagus and airway.

This important role of Hedgehog signaling in normal esoph-ageal development was confirmed in mice with mutations inthe Gli transcription factors. In Gli2/ mice, the esophagushas a very small lumen surrounded by a poorly developedmesenchymal layer that fails to develop an �Sma-positivesmooth muscle layer. In Gli2/ Gli3�/-mutant mice, theforegut is hypoplastic and fails to form the appendages for thetrachea and lungs at E9.5 (58). In Gli2/ Gli3�/ mice thatsurvive until later stages of development only a very smallproximal esophageal remnant can be observed and the micelack both trachea and lungs.

The striking similarity between Shh- and Sox2-mutant micesuggests that these factors are functionally related. It is un-likely that Sox2 acts directly downstream of Shh because Shhsignaling is uniquely to the mesenchyme. There are two alter-native options that are not mutually exclusive. First, endoder-mally expressed Sox2 could act upstream of Shh as a criticaltranscription factor required for Shh expression. Alternatively,endodermal Sox2 expression could depend on mesenchymallyexpressed factors that are controlled by Shh signaling.

The transcription factor Foxf1 is probably the key mesen-chymal target of Shh signaling. Mahlapuu and colleagues (46)have demonstrated that mesenchymal expression of Foxf1 canbe induced by ectopic expression of Shh and that Shh-mutantmice lack mesenchymal Foxf1 expression in the foregut. TheFoxf1 homozygous mutation leads to early embryonal lethalityand cannot be evaluated for a foregut phenotype. However,Foxf1�/-mutant mice have a clear foregut phenotype that isvery similar to Shh- and Gli-mutant mice. In Foxf1�/ mice theesophagus is poorly developed and fails to separate properlyfrom the trachea, the lungs are hypoplastic, and branchingmorphogenesis is reduced (46). Thus Sox2/Shh signaling in-duces mesenchymal Foxf1 expression, which is required toallow the mesenchymal cells to support esophageal elongationand survival and the appropriate separation of the esophagusand airways.

Bone morphogenetic protein signaling. Members of thebone morphogenetic protein (Bmp) signaling pathway play acritical role in foregut development. Bmp4 and Bmp7 are themajor Bmp ligands expressed during foregut development (40,72). These Bmps display a nonoverlapping expression pattern,with expression of Bmp4 being restricted to the mesenchymeventral to the developing trachea and Bmp7 expression in theepithelium of the developing esophagus and its surroundingposterior mesenchyme. Given the strong similarity betweenBmp4 mutant and Bmp receptor-mutant mice (see below), itseems that the mesenchymally expressed Bmp4 is the key Bmpligand during gut-airway development. Signaling by the Bmppathway seems to occur mainly in the posterior foregut endo-derm and mesenchyme. This was assessed by LacZ staining ina BRE-LacZ Bmp signaling reporter mouse (72) and immuno-histochemical localization of the phosphorylated form ofSmads1, 5, and 8 (40), the Smads that mediate Bmp signaling.

Consistent with expression of Bmp4 in the ventral meso-derm surrounding the developing trachea, Bmp4 plays a criti-cal role in tracheal development. At E11.5 Bmp4 conditionalknockout animals in which Bmp4 is deleted from the foregut

endoderm and mesoderm show a clear failure of foregutseparation with a single tube connecting the pharynx to thestomach and hypoplastic lungs (40). This tube shows esopha-geal type differentiation since it is positive for esophagealendodermal marker Pax9, negative for the tracheal endodermmarker Nkx2.1, and negative for the tracheal mesenchymemarker Col2a. The authors of this study found that Bmp4signaling is not required for specification of the tracheal pri-mordium, which formed normally at E9.25. However, thetracheal primordium was reduced in size compared with wild-type mice at E9.5. Thus Bmp4 is required for proper airwaydevelopment after the initial specification of the tracheal pri-mordium. The importance of Bmp4 in airway developmentmay explain why the airways fail to develop properly inShh-mutant mice since Bmp4 is one of the key mesenchymaltargets of Hedgehog-Foxf1/Foxf2 signaling in the esophagus(42) and the intestine (11).

Mice in which both Bmp receptor 1a and 1b were specifi-cally deleted from the endoderm by using a ShhCre showed asimilar phenotype as the Bmp4 conditional-mutant mice. TheBmpr1a/b double-mutant mice developed a single foregut tubethat was positive for esophageal endodermal marker Sox2 andnegative for the airway endodermal marker Nkx2.1, thus againshowing that Bmp signaling is required to induce and/ormaintain a specific airway endodermal phenotype.

Conversely, actively antagonizing Bmp signaling has alsobeen shown to play a role in protecting the esophageal endo-derm against the airway phenotype inducing influence of theBmp signaling pathway. Several Bmp antagonists are ex-pressed in the developing foregut (72). Of those, Noggin islikely the most relevant Bmp antagonist during foregut devel-opment, since Noggin-mutant mice have a clear foregut phe-notype (41, 68). Noggin is expressed in the dorsal foregutendoderm and lung mesenchyme from E10.5–11.5 and at laterstages (E14.5) is confined to the developing esophageal smoothmuscle layer. Loss of Noggin expression in Nog/-mutantmice showed an opposite phenotype of the Bmp4 and Bmpr1a/b-mutant mice with intact airway differentiation in a singleforegut tube connecting the airways to the stomach (68). Thissuggests not only that Bmp signaling is required for properairway differentiation but that suppression of Bmp signaling inthe posterior foregut is equally important to allow properesophageal development. The reciprocal nature of Noggin andBmp4 signaling was clearly demonstrated by the fact that thephenotype of the Nog/ mice could be rescued by reducingthe gene dose of Bmp4 in Nog/Bmp4�/-mutant mice (68).The importance of the reciprocal regulation of Bmp signalingbetween airway and esophagus was explained with an elegantexperiment by Domyan et al. (18) since these authors showed thatthe Bmpr1a/b airway phenotype can be rescued by deletion ofSox2 in Bmpr1a/b Sox2 double-mutant animals. Domyan andcolleagues found that Bmp signaling directly represses theSox2 promoter (18). Thus Bmp signaling in the airway endo-derm is required to repress Sox2 expression, allowing theairway endodermal phenotype to be expressed. Conversely,Noggin-mediated repression of Bmp signaling allows theproper development of the esophageal endoderm by protectingSox2 expression against the repressive influence of the Bmppathway. In conclusion, an endodermal-mesenchymal signal-ing network has been discovered in which endodermally ex-pressed Shh induces Bmp4 in the mesenchyme via the Foxf1

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transcription factor. This Bmp4 signals reciprocally to theendoderm of the developing airways to repress endodermalSox2 expression and allow proper airway differentiation. Theesophageal endoderm is protected from this mesenchymalBmp4 signal by secreting the Bmp antagonist Noggin.

Fibroblast growth factor signaling. The fibroblast growthfactors (Fgfs) are a large group of morphogens with an impor-tant role in endodermal development. During gut-airway de-velopment this pathway provides a critical signal from themesenchyme to the overlying endoderm. Fgfs act throughtyrosine kinase transmembrane receptors. Thus far, four Fgfreceptors have been identified. The tissue-specific alternativesplicing of the FGF receptors I–III is the main mechanismby which FGF-FGFR binding specificity is regulated (61).This splicing event gives rise to epithelial “b” isoforms(FGFRIb to FGFRIIIb) and mesenchymal “c” isoforms (FG-FRIc to FGFRIIIc), which differ in the binding specificityprofiles for the many different Fgfs. The key Fgf with anestablished role in foregut development is Fgf10. At E10.5Fgf10 is expressed in the anterior mesenchyme surrounding theprospective trachea. The importance of this mesenchymalFgf10 expression is underscored by the fact that Fgf10-mutantmice develop a trachea but completely lack further develop-ment of the lung buds (55, 76). Fgf10 in the mesenchymesignals to a specific IIIb isoform of the Fgfr2 (the main receptorfor Fgf10) expressed in the epithelium. Indeed, mice thatspecifically lack the Fgfr2 IIIb isoform display lung agenesissimilar to Fgf10-mutant mice (16). The role of mesenchymal-to-epithelial Fgf10-Fgfr2 IIIb signaling seems to lie in thereciprocal regulation of Nkx2.1/Sox2 expression. Fgf10 pro-motes an airway phenotype by positively regulating Nkx2.1expression and repression of the expression of Sox2 (69). It hasnot been examined how the mesenchymal-endodermal Fgf10-Fgfr2 IIIb signaling axis relates to the Shh-Bmp4 signalinginteractions in the foregut that have been mentioned above.

Wnt signaling. Similar to the Bmps and Fgfs, Wnts play akey role in airway development. Wnt2 and Wnt2b are ex-pressed in the ventral mesoderm that surrounds the endodermof the prospective airways around E9.0–10.5 (24). Wnt7b isexpressed in the ventral endoderm at the same time in devel-opment (77). Wnt2/2b double-mutant mice fail to induce ex-pression of Nkx2.1 and display complete lung and trachealagenesis with intact esophageal development (24). Wnt2b mu-tant embryos show a much less dramatic phenotype withmodest lung hypoplasia. Airway development requires canonicalWnt signaling because Shh-Cre-Ctnnb1fl/fl-mutant mice in which-catenin is specifically deleted from the early endoderm are aphenocopy of Wnt2/2b-mutant mice (24). The esophagus devel-ops normally in Shh-Cre-Ctnnb1fl/fl mutants, indicating that ca-nonical Wnt signaling is not involved in normal esophagealdevelopment (24). In Shh-Cre-Ctnnb1(ex3)fl/wt mice, in which-catenin is constitutively activated in the early endoderm, aninduction of Nkx2.1-positive cells is observed in the develop-ing esophagus with concomitant loss of p63 expression. Thisfirmly establishes the important role of canonical Wnt signal-ing in airway epithelial specification. One of the transcriptionfactors that is required to repress Wnt signaling in the devel-oping esophagus to allow normal esophageal development isBarx1 (97). Barx1 is expressed in the mesoderm in between thedeveloping esophagus and trachea and it has been suggestedthat Barx1 negatively regulates Wnt signaling through the

regulation of secreted frizzled-related proteins (97). BecauseWnt2/2b-mutant mice showed complete loss of Fgf10 expres-sion it seems that canonical Wnt acts upstream of Fgf10 andNkx2.1 as one of the key drivers of airway epithelial specifi-cation.

In conclusion, the tissue-specific transcription factors andmorphogenetic pathways that regulate esophagus-airway sep-aration and differentiation have partially been resolved. Sox2and Nkx2.1 are the key endodermal transcriptional regulatorsof esophageal and airway fate, respectively. Development ofthe esophagus critically depends on Hedgehog signaling andactively suppressing BMP signaling. In contrast, the Fgf, Bmp,and Wnt signaling pathways are key regulators of airwaydevelopment (summarized in Fig. 3).

ESOPHAGEAL EPITHELIAL HOMEOSTASIS IN THE ADULT

Murine Esophageal Epithelium

In contrast to the rest of the gastrointestinal tract, which iscovered with a single layer of columnar epithelium, the esoph-agus is lined with a multilayered squamous epithelium (Fig. 2).This epithelial phenotype is reflective of its role to transportrather than modify and absorb luminal content. The esophagealepithelium in the mouse is constantly renewed from a popula-tion of cells that are neatly organized with their nuclei perpen-dicular to the basement membrane, the so-called basal layer(37, 49). As differentiating cells leave the basal layer, theychange their shape and orientation to become larger, flattened,and aligned parallel to the basement membrane. These cellshave a large cytoplasm, causing the enlarged nuclei to bespread further apart from each other, compared with the nucleiin the basal layer. Advancing upward, toward the lumen, nucleiare degraded and cells develop keratohyalin granules, whichcan be identified as basophilic small round structures. Thesurface layer of the murine esophagus is keratinized, possiblyto form a strong protective layer against abrasive food com-ponents. The rate of proliferation of the cells in the basal layeris tightly coupled to the rate at which differentiating cells arelost in the esophageal lumen. The mechanisms that regulatehomeostasis in this dynamic equilibrium have not been de-scribed.

Keratins as Markers of Esophageal Epithelial Differentiation

Keratins are the building blocks of intermediate filamentsthat form part of the cells cytoskeleton (34). Keratins areexpressed in a highly cell-type- and maturation-state-specificmanner, and several keratins are useful markers of the differ-entiation state of esophageal epithelial cells. In esophagealbasal cells three keratins are present. Keratin 14 is paired withkeratin 5 (96), and both are expressed in all basal layer cells inthe adult squamous epithelium. Keratin 15 is the third keratinmember expressed specifically in the basal layer (94). Asesophageal epithelial cells leave the basal layer and start todifferentiate they shut down expression of keratins 5, 14, and15 and induce the expression of keratin 4 and its partner keratin13 (90). Differentiating cells start to degrade their nucleus andother organelles and make keratohyalin granules, which con-tain profillagrin (45, 47). This is the precursor to fillagrin (33),which will aggregate keratins into tight bundles, resulting inthe typical flattened shape of differentiated esophageal epithe-

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lial cells. In addition, cells will start to synthesize specializedproteins such as involucrin (3) and loricrin (51), which formthe cornified cell envelope just beneath the plasma membrane,a structure with a key role in epithelial barrier formation inkeratinized epithelia.

Human vs. Murine Esophagus

Most studies on dynamics of epithelial homeostasis areperformed in rodents. It is, however, important to note that

there are key differences between the murine and humanesophageal epithelium (Fig. 4). First of all, the human esoph-ageal epithelium contains more cell layers and it is foldedalong papillae. Proliferation and mitosis in the mouse is limitedto basal cells (37, 49). In humans this is extended to the fifth tosixth suprabasal layers (4). Unlike the murine esophagealepithelium, the human esophageal epithelium is nonkeratinizedand cells retain their nucleus (27). Therefore, keratohyalingranules are rare. In rodents, keratinization of the esophageal

Fig. 3. Signaling pathways controlling esophageal development and epithelial homeostasis. Schematic overview of the major (morphogenic) signaling pathwaysinvolved in the development of the airway (tracheal) and esophageal endo- and mesoderm (A) and in homeostasis of the adult esophageal epithelium (B). Fora more detailed description of the pathway components involved, see main text. scFz, secreted Frizzled (Wnt antagonist); Ng, Noggin; Fst, Follistatin; Grem2,Gremlin2 [all bone morphogenetic protein (BMP) antagonists].

Fig. 4. Adult murine esophageal epithelium vs. adult human esophageal epithelium. Schematic representation of the tissue architecture of the esophagus toindicate the differences between mouse and human esophageal epithelium.

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epithelium may serve to protect against abrasive dietary com-ponents. The human esophageal epithelium is exposed toharmful dietary substances as well. The main mechanisms bywhich the esophageal epithelium copes with this is high turn-over of epithelial cells. Esophageal submucosal glands (44) arepresent in human but not in mice and may play an importantprotective role in humans.

Regulators of Esophageal Epithelial Proliferation andDifferentiation

The mechanisms by which esophageal epithelial homeosta-sis is regulated are relatively poorly characterized. It is becom-ing clear that many of the same pathways that regulate mor-phogenesis of an epithelium during development are often alsocritical to regulate epithelial homeostasis in the adult epithe-lium (see Fig. 3) (88). This notion seems to be valid for theesophageal epithelium. For example, as described above, Sox2is the key tissue-specific transcriptional regulator that definesthe esophageal epithelial phenotype during development. In theadult epithelium Sox2 is expressed in virtually all cells of thebasal layer. Lineage tracing of Sox2� cells in the adultesophageal epithelium showed that Sox2� cells can generatelong-lived clones of cells that persist in the esophageal epithe-lium (1). In transgenic mice that express the thymidine kinasegene under control of the Sox2 promoter, treatment withganciclovir causes ablation of Sox2-expressing cells and thisresults in complete loss of basal cells (1). Reciprocally, over-expression of Sox2 leads to an increase in epithelial progenitorcells and loss of differentiated features (43). Together thesedata support the notion that the key role of Sox2 as a tissue-specific transcription factor in development is maintained inthe adult esophageal epithelium.

A second transcriptional regulator with a conserved rolebetween development and adult epithelial homeostasis may bep63. Work in esophageal squamous cell carcinoma (ESCC)cell lines suggests that p63 may be required for epithelialproliferation (85). Although the in vivo role of p63 in adultesophageal epithelium has not been addressed, genetic deletionof p63 in organotypic culture derived from adult esophagealepithelium indeed inhibited their self-renewal (30).

Since Shh is a morphogen with a key role in esophagealmorphogenesis, we and others have examined the role of Shhsignaling in the adult esophagus (29, 89). Shh is expressed byepithelial cells of the basal layer in the adult esophagus. Incontrast to the exclusively paracrine Hedgehog signaling thecuboidal epithelium during development, we found thatHedgehog signaling is also autocrine in the adult squamousepithelium (similar to the skin). Using in situ hybridization wefound that the cells in the basal layer expressed both theHedgehog receptor Smo and transcription factor Gli-1 andbasal cells were marked by LacZ expression in Gli1-LacZreporter mice (89). We examined the role of Shh by activatingthe Hedgehog (Hh) pathway using two mouse models: one inwhich the inhibitory receptor Ptch1 can be conditionally de-leted and another in which Hh pathway transcription factorGli1 can be conditionally overexpressed. In these mouse mod-els, which both lead to increased Hh signaling, we observed anexpansion of the proliferating cell compartment accompaniedwith impaired maturation and migration of epithelial cells. Thisis consistent with an autocrine role for Shh signaling in the

epithelial cells of the basal layer of the adult esophagus andindicates that Hh signaling regulates the phenotype of basalcells in the esophageal epithelium and promotes their prolifer-ation (89).

Recently it was reported that BMP signaling (and antago-nism) is also involved in homeostasis of the adult esophagealepithelium (31). In contrast to their nonoverlapping expressionin development, in the adult mouse esophagus, both BMP4 and7 are expressed in the epithelial basal layer and signal towardthe suprabasal epithelium. Simultaneously, the expression ofthe BMP antagonists follistatin and gremlin2 limit BMP sig-naling in the basal layer itself and the underlining mesen-chyme, respectively. Genetic hyperactivation of BMP signal-ing via epithelial overexpression of a constitutively activeBMP receptor 1 results in large-scale differentiation of esoph-ageal epithelial progenitor cells (31).

In addition to the factors shown to be important in develop-ment, several other signaling molecules and mechanisms havebeen described to regulate adult esophageal homeostasis:

Two transcription factors of the Krüppel-like factor (Klf)family also play a role in homeostasis of the esophagealepithelium in the adult animal. Expression of Klf5 is restrictedto the basal layer and seems to regulate proliferative capacity.Transgenic overexpression of Klf5 in the esophageal epithe-lium results in a twofold increase in proliferation rate, withoutfurther abnormalities in esophageal epithelial homeostasis(23). In contrast to Klf5, Klf4 is expressed in the suprabasallayer. Klf4 plays a critical role in normal esophageal epi-thelial differentiation. Klf4-deficient mice show impaireddifferentiation and hyperproliferation, resulting in epithelialdysplasia (84).

One of the major cell-to-cell signaling pathways with a rolein esophageal homeostasis is the Notch signaling pathway.Ohashi et al. (59) have shown that Notch signaling through thetranscription factor CSL is required for human esophagealepithelial differentiation in organotypic cultures in vitro and inthe mouse esophageal epithelium in vivo. Their work sug-gested a key role for the expression and activation of NOTCH1and NOTCH3. The key role for Notch signaling in esophagealhomeostasis is underscored by the finding that Notch pathwaygenes are frequently mutated in esophageal squamous cellcarcinomas (21).

Interestingly, we found upregulation of Notch pathway com-ponents (Dll3, Jag2, and Hes5) in a mouse model that leads toesophageal precursor cell differentiation (73). In this model wechemically induced endoplasmic reticulum (ER) stress andsubsequent unfolded protein response (UPR) activation viathapsigargin treatment. Thapsigargin is a plant-derived inhib-itor of ER Ca2�-ATPases that induces ER stress by Ca2�

-depletion of the ER. In vivo, thapsigargin treatment led toreduced proliferation and increased progenitor differentiationin esophageal epithelium and correlated with increased expres-sion of several Notch signaling components. More evidence forthe involvement of the UPR in esophageal epithelial homeo-stasis came from experiments with a genetic ER stress model,Ah1Cre-Rosa26-LacZ-Grp78/ mice (73). UPR activation inresponse to conditional deletion of a major ER chaperoneGrp78 in esophageal epithelium of these mice resulted in rapiddifferentiation followed by repopulation of the epithelium fromthe nonrecombined wild-type cells, as was found in intestinalepithelium (25). This suggests that also in the esophageal

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epithelium the UPR may serve as a quality control mechanismthat forces progenitor cells with accumulated unfolded proteinsto initiate differentiation.

Esophageal Epithelial Pathophysiology, the Development ofBarrett’s Esophagus

Although the focus of this review is on the pathways thatmaintain esophageal epithelial homeostasis and we have lim-ited insight into how these pathways are deregulated duringcarcinogenesis, we will briefly review the available data onhow the signaling pathways discussed above have been impli-cated in the development of Barrett’s esophagus, the precursorto esophageal adenocarcinoma (64, 74). For a broader reviewon Barrett’s esophagus we refer to recently published reviews(15, 80).

The development of esophageal adenocarcinoma occurs in astepwise manner that is relatively well characterized. Themajor risk factor for the development of esophageal adenocar-cinoma is gastroesophageal reflux disease (GERD). In GERD,the lower esophageal sphincter fails to prevent reflux of gastriccontent, thereby leading to exposure of the esophageal epithe-lium to gastric acid and bile acids. This can eventually lead tothe development of chronic esophageal inflammation and ul-ceration. In a subset of patients with esophagitis, the stratifiedsquamous epithelium at the gastroesophageal junction is con-verted to intestinal-type columnar epithelium, a process calledmetaplasia. This intestinal metaplasia is called Barrett’s esoph-agus and is a precursor lesion for the development of adeno-carcinoma. Population-based studies have shown that Barrett’sesophagus is present in 0.5–1.5% of the Western population(15). The progression of Barrett’s epithelium to adenocarci-noma again occurs in a well-established stepwise fashion inwhich the epithelium will first develop areas of low-gradedysplasia, which progresses to high-grade dysplasia and can-cer. The risk of developing esophageal adenocarcinoma is verylow [between 0.05–0.5% per year for nondysplastic metaplasia(15)] but increases steeply as the epithelium progresses fromnormal epithelium to low-grade and high-grade dysplasia.

The molecular mechanism of the conversion of normal esoph-ageal epithelium to intestinal metaplasia is incompletely under-stood and it is not known whether the metaplastic epithelium isderived from the normal esophageal epithelium or arises fromgastric cardia stem cells (66) or from remnant embryonic epithe-lial cells that persist at the gastroesophageal junction, as has alsobeen proposed (93). Below we will discuss some of the pathwaysthat may play a role in the development of Barrett’s esophagus.

Genetic predisposition. Genomewide association studies(GWAS) that examined genetic predisposition to the develop-ment of Barrett’s esophagus have implicated several pathwayswith a role in the development of the esophagus (38, 62, 81).One of the most significant associations with the developmentof Barrett’s esophagus found in the first large GWAS is asingle-nucleotide polymorphism (SNP) that is very close toFOXF1 (81), the mesenchymally expressed Hedgehog targetthat acts upstream of BMP4 during development (see above).Although, it was described that several transcription factorsthat regulate expression of FOXF1 bind in the region that is inlinkage disequilibrium with the associated SNP, it is not yetclear how the risk allele affects FOXF1 expression. Given therole of BMP4 in the development of Barrett’s esophagus

described below it could be speculated that the FOXF1 riskallele may act to increase stromal BMP4 expression; however,this has not yet been investigated.

A subsequent large GWAS identified a further two transcrip-tional regulators with a key role in development in the risk todevelop Barrett’s esophagus (38). The first is FOXP1. Foxp1 isexpressed by the esophageal epithelium and muscle layerduring development (78) and it has been shown that Foxp1cooperates with Foxp2 in esophageal development. Foxp2/

Foxp1�/ double mutant had a defect in the development ofthe esophageal muscle wall. However, the role of Foxp1 in theesophageal epithelium is not known and it is not known whichof the morphogenetic pathways discussed above interact withFOXP1 in the context of the esophagus. Intriguingly, however,FOXP1 is overexpressed in diffuse large B cell lymphoma,where it enhances Wnt signaling (91). Increased Wnt signalingcould play a role in the development of intestinal metaplasiasince Wnt signaling needs to be repressed to allow the devel-opment of normal esophageal epithelium and the Wnt pathwayis the major pathway in the specification of the intestinal stemcell phenotype. The potential for interaction with the Wntsignaling pathway is something FOXP1 has in common withthe second transcription factor with an association with Bar-rett’s esophagus identified in the same GWAS (38).

BARX1. BARX1 is an important repressor of the Wntsignaling pathway during development (discussed above). Inaddition, TBX5, a transcription factor that was identified in thelargest GWAS to date (62), is a key regulator of limb devel-opment through the regulation of the expression of Wnt ligands(83). The third GWAS also identified a locus near GDF7, amember of the BMP pathway that is interesting in the light ofthe potential role of BMP signaling in the development ofBarrett’s esophagus discussed below. In conclusion, although itis not always easy to interpret the data from a GWAS and thefunctional consequences of the different alleles that have nowbeen associated with Barrett’s have yet to be elucidated, itseems that most factors have a likely association with the Wntsignaling pathway, a key pathway for intestinal stem cellspecification, or the BMP pathway, which has also been im-plicated in functional experiments discussed below.

Functional experiments. It has been shown that Barrett’sepithelium gains the expression of key transcriptional regula-tors of intestinal epithelial cell fate such as CDX1 and CDX2,and it has been reported that both of these factors are inducedby acid and bile (80). Transgenic ectopic expression of Cdx2can convert gastric epithelium to intestinal metaplasia in mice(79). However, a similar approach has failed to induce meta-plasia in the esophagus (35), indicating that expression ofCDX2 alone is insufficient to cause Barrett’s esophagus. Oneof the factors that could play a role in the development ofmetaplasias in general is the ectopic and aberrant expression ofmorphogens in immune cells and/or mesenchymal cells due tothe chronic inflammatory infiltrate. Indeed, in Barrett’s esoph-agus it was found that BMP4 is overexpressed in the stromaand that treatment of esophageal cells with BMP4 inducescolumnar morphology (54). This suggests that stromal BMP4expression may play an important role in the development ofintestinal metaplasia. However, treatment with BMP4 alonewas insufficient to cause intestinal metaplasia (54). Otherinvestigators have later found that Hedgehog ligands Shh andIhh are overexpressed in Barrett’s esophagus vs. normal epi-

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thelium and that epithelial mesenchymal Hedgehog signalingmay be one of the factors driving stromal BMP4 expression(92). The potential role of BMP signaling in metaplasia is anintriguing finding in the light of our understanding of themechanism of esophageal epithelial development. As dis-cussed above, experiments in mice have established that it iscritical to normal esophageal development that the epitheliumis protected against BMP signaling by the expression of BMPantagonists (see Bone morphogenetic protein signaling sectionabove). It has recently been elegantly shown that a combina-tion of BMP4 signaling with CDX2 expression is sufficient toinduce intestinal metaplasia in esophageal cells, suggestingthat these two factors combined may play a critical role in theprocess (48).

Regarding the factors in the inflammatory infiltrate that pro-mote the development of metaplasia and dysplasia in the intestinaltype epithelium, important recent work has shown that Il-1 andIl-6 play a key role in this process and both cytokines can inducethe expression of Shh as well as Bmp4 in a mouse model (66).

In conclusion, although much remains to be investigated re-garding the molecular mechanism of the development of Barrett’sesophagus, it seems that induction of expression of CDX1/2 bygastric content in combination with epithelial expression ofHedgehogs and stromal overexpression of BMP4 due to thechronic exposure to Il-1 and Il-6 are important contributors.

Esophageal Stem Cells

In concordance with both the skin epidermis and the gastro-intestinal tract, the esophageal epithelium is constantly re-newed. This suggests the presence of an actively proliferatingstem cell population to fuel this renewal. However, despite thefact that in the last decade stem cells have been identified in themouse skin and the other tissues of the murine gastrointestinaltract (6, 7, 22, 67), to date no esophageal stem cell has beenconclusively demonstrated.

Currently, there is a lack of consensus about the presence ofdedicated stem cells in the esophagus. The mouse esophagealepithelium is devoid of clearly identifiable structural featuressuch as crypts and glands that serve as stem cell niches in othertissues. However, the region to which proliferation is restrictedhas been unequivocally pinpointed. Pioneering work performedby Leblond and colleagues (37, 49) in the rat esophagus showedwith the use of [3H]thymidine pulse-chase experiments that pro-liferation is restricted to the basal layer. This led the authors toconclude that “if stem cells are defined as cells which producecells similar to themselves as well as differentiating cells, the basalcells are the stem cells of the esophageal epithelium” (37).

The current debate focuses on the question whether indeedall basal cells have an equal capacity for self-renewal (5) orwhether the basal layer is organized in a stem cell-transitamplifying (TA) cell hierarchy (32) as is found in for examplethe small intestine. In the following paragraphs we will try tosummarize the evidence presented for the two hypotheses. Wewill restrict ourselves to the potential presence of stem cellpopulation (s) during homeostasis since even less is knownabout the behavior of potential stem cells during wound repairand carcinogenesis.

To avoid confusion in nomenclature we will use a descrip-tion to define a dedicated tissue stem cell as follows: atissue-specific stem cell has the ability to generate new stem

cells, i.e., has self-renewal capacity, and is capable of gener-ating all the cell types present in a tissue, i.e., has tissue-renewal capacity. Of note, these characteristics are, in ouropinion, independent of the cycling time and/or label-retainingcharacteristics of these cells. It should be mentioned here thatthe well-characterized Lgr5� stem cells of the small intestinecycle once every 24 h and can be considered fast cycling (7). Incontrast, the term stem cell has also been used for slow-cycling,“label-retaining cells” (LRCs) that contribute to epithelial homeo-stasis after damage (39). These LRCs are in general cycling onlyrarely in a homeostatic tissue and can therefore be identified bythe prolonged retention of a DNA label after chronic infusion ofsuch a label. With an elegant in vivo Histone2B-GFP pulse chaselabeling experiment, Doupe et al. (19) showed that there are lownumbers of LRCs found in the esophagus basal layer epithelium,as was previously described (32). However, Doupe et al. foundthat these cells are not of epithelial origin since these cellsexpressed the hematopoietic lineage marker CD45. This suggeststhat a population of label-retaining stem cells may indeed beabsent from the murine esophageal epithelium. In humans such aLRC might be present, since one study described the identificationof LRCs after 5-iodo-2=-deoxyuridine labeling in patients under-going esophagectomy (63).

Importantly, the presence (or absence) of a pool of labelretaining (reserve stem) cells does not exclude the existence offaster-cycling dedicated stem cells in the esophagus. To furtheraddress this question, Doupe et al. (19) examined the fate ofsingle-cell-derived clones in the basal layer in a lineage traceexperiment, using a Ah-CreERT2*LSL-eYFP-reporter mouse.Mathematical modeling of the results led them to conclude thatthe majority (65%) of the basal layer consists of esophagealprogenitors (EPs). These EPs divide on average �2 /wk andfor every division “esophageal progenitors are functionallyequivalent” (19). This concept is very similar to the interpre-tation of previous research by Leblond and colleagues (49).

In contrast, other studies suggest that the mouse basal layeris functionally heterogeneous and provide evidence for a stemcell/TA hierarchy in the esophageal epithelium. Kalabis et al.(32) isolated a subpopulation of cells from the esophagealepithelium on the basis of a dye-exclusion method and pro-vided evidence for high clonogenic potential within this sub-population of cells, suggesting that they might be stem cells.

In addition, several other studies have used different cellsurface markers to isolate distinct subpopulations from themurine (17, 30, 32) and also human (4, 30, 60, 75) esophagealepithelium via FACS sorting. With one exception (4), theinvestigators report considerable differences in self-renewalcapacity between the different epithelial populations. Thesedata provide compelling evidence for the existence of pheno-typically and functionally distinct epithelial cell populationswithin the basal layer of mice and humans, suggestive of amore hierarchical stem cell-transit amplifying cell organizationsimilar to most other epithelial tissues.

Additionally, methods have now been established for bothmouse and human 3D organotypic esophageal epithelial cul-tures (17, 30, 69). Culturing of such esophagospheres willprovide an easy platform to advance our insights into the regu-lation of the esophageal stem/progenitor cell populations andepithelial homeostasis. In addition, such cultures can be used tomodel esophageal carcinogenesis, similar to the organoid culturesthat are being used to model colorectal carcinogenesis (50, 86). In

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summary, the existence of a specific esophageal stem cell popu-lation is still a matter of debate although several lines of evidenceindicate that there may be considerable heterogeneity of self-renewal potential in the basal layer of the esophagus. Further useof the established tools to culture primary esophageal cell in vitroand lineage tracing in vivo will be required to shed more light onthis important issue.

CONCLUDING REMARKS

Clearly we still have very limited insight in pathways and genesinvolved in normal homeostasis of the esophageal epithelium.Enhancing our knowledge about the mechanism of proliferationand pathways driving differentiation is therefore crucial. It wouldlead to better understanding of the mechanisms involved in tissuerepair, the development of Barrett’s esophagus, and carcinogen-esis. The existence of specific stem cell populations that driveesophageal renewal needs further experimental evidence, espe-cially given an importance of stem cells as the most likely cell oforigin during oncogenic transformation.

GRANTS

G. van den Brink acknowledges financial support from the NetherlandsOrganization of Scientific Research (NWO; VIDI Grant).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author (s).

AUTHOR CONTRIBUTIONS

S.L.R. and B.B. prepared figures; S.L.R., B.B., V.M., and G.R.v.d.B.drafted manuscript; S.L.R., B.B., V.M., and G.R.v.d.B. edited and revisedmanuscript; S.L.R., B.B., V.M., and G.R.v.d.B. approved final version ofmanuscript; G.R.v.d.B. conception and design of research.

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