organog timo ann review imm 08

ANRV338-IY26-12 ARI 16 February 2008 12:33

Thymus OrganogenesisHans-Reimer RodewaldInstitute for Immunology, University of Ulm, D-89070 Ulm, Germany;email: [email protected]

Annu. Rev. Immunol. 2008. 26:35588

First published online as a Review in Advance onNovember 30, 2007

The Annual Review of Immunology is online atimmunol.annualreviews.org

This articles doi:10.1146/annurev.immunol.26.021607.090408

Copyright c 2008 by Annual Reviews.All rights reserved

0732-0582/08/0423-0355$20.00

Key Words

thymus medulla, thymus cortex, germ layers, thymus epithelialstem and progenitor cells, transcription factors, cervical thymus,reaggregate organ cultures, Cre recombinase, fate mapping, nudeblastocyst complementation

AbstractThe epithelial architecture of the thymus fosters growth, differen-tiation, and T cell receptor repertoire selection of large numbers ofimmature T cells that continuously feed the mature peripheral T cellpool. Failure to build or to maintain a proper thymus structure canlead to defects ranging from immunodeciency to autoimmunity.There has been long-standing interest in unraveling the cellular andmolecular basis of thymus organogenesis. Earlier studies gave im-portant morphological clues on thymus development. More recentcell biological and genetic approaches yielded new and conclusiveinsights regarding the germ layer origin of the epithelium and thecomposition of the medulla as a mosaic of clonally derived islets. Theexistence of epithelial progenitors common for cortex and medullawith the capacity for forming functional thymus after birth has beenuncovered. In addition to the thymus in the chest, mice can have acervical thymus that is small, but functional, and produces T cellsonly after birth. It will be important to elucidate the pathways fromputative thymus stem cells to mature thymus epithelial cells, andthe properties and regulation of these pathways from ontogeny tothymus involution.

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INTRODUCTION

The classical embryological view of thymusdevelopment that had been perpetuated fordecades has undergone major revisions withinthe past few years. Advancing the under-standing of thymus organ development hasbeen slow by comparison to the progressin research addressing cellular pathways andunderlying molecular mechanisms of devel-oping T cells. Why are experiments address-ing the development of the thymus inter-esting? The unique function of the thymusin establishment and maintenance of the Tcell arm of the immune system is intimatelylinked to specialized functions of thymus stro-mal cells and the thymus architecture (1, 2).These cells are of major immunological rele-vance for the intrathymic selection of a self-tolerant and self-MHC-restricted T cell anti-gen receptor (TCR) repertoire (36). It isbroadly recognized that thymus structure iskey to these specic immunological propertiesof the thymus. This, combined with the real-ization that thymus structure and its develop-ment are, in large part, still uncharted terri-tory, has spurred increased interest in thymusorganogenesis.

Novel experimental access has been facil-itated by the development of experimentaltools such as, to name a few, stromal cell isola-tion by phenotype-based cell sorting (e.g., 79), dissociation and reaggregation of stromalcell subsets to probe their functional capac-ity in vitro (10, 11) and in vivo (e.g., 1215),or global gene expression analyses to deter-mine the pattern of self-antigen expression inthymus epithelial cell (TEC) subsets (5). Keyquestions in thymus organogenesis surroundthe fundamental cellular and molecular mech-anisms that lead to the formation of a normalthymus. Areas of interest include the tran-sient or permanent germ layer contribution tothymus structure, that is, origin of TEC, andother stromal elements; the quest for thymus-building and/or maintaining stem or progen-itor cells; an understanding of the turnover ofthymus stromal elements in the steady-state

thymus; and the role of thymus stromal cellsin thymus involution or pathology-associatedtransient thymus malfunctions (16). In par-ticular, quantitative answers to many of thesequestions would be useful. For instance, thenumber of cells with TEC-forming potential(TEC progenitors) needs to be determined,as well as their clonogenic potential, steps ofcommitment, life span, etc.

Thymus stroma can be viewed as all non-hematopoietic components of the thymus thatare functionally dened as those elements, re-gardless of their origin and lineage, that con-stitute the thymus structure, and hence pro-vide the matrix on which thymocytes develop(Figure 1). A simple, but useful classica-tion of stroma lacking the pan-hematopoieticmarker CD45 is based on keratin expression,in that keratin+ cells represent thymus epithe-lium, and keratin cells are a mixture of mes-enchymal cells. Keratin+ cells are composedof two major subsets referred to as corticalTEC (cTEC) and medullary TEC (mTEC).Keratin cellsby default collectively con-sidered as mesenchymal cellsinclude bro-blasts (17), nonbroblastic mesenchymal cells(9), capsule- and septae-forming connectivetissue cells, and endothelial cells forming thetypical thymus vasculature (9, 18, 19). Fi-nally, dendritic cells and macrophages that areCD45+ hematopoietic cells are also importantelements of thymus stroma.

Stroma is not only heterogeneous at agiven time point, but its composition alsovaries considerably over time (20). Such re-structuring can reach extreme forms un-der thymus-ablating conditions (steroid treat-ment, irradiation, cachectic conditions) orwith age-associated thymus involution, eventsthat seriously impair thymus function (16).The cellular heterogeneity of thymus struc-ture, as is true for most organs, poses in-trinsic difculties to analyze the developmentor function of given cell types in the phys-iological context and their transient or per-manent contribution to the thymus structureand function. Moreover, direct from indirect

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Thymus

CD45+

CD45

Keratin

Capsule

Septae

Endothelium

Fibrobasts

Nonfibroblast- mesenchyme

Thymocytes

Dendritic cells

Macrophages

B cells

Hematopoietic stem cells;

continuous colonization

Third pharyngeal pouch

endoderm TEC progenitors;

Foxn1-dependent

Phenotypes Cell types

Neural crest mesenchyme

early in ontogeny with

minor contribution to adult

thymus?; replenished from

local mesenchyme?

Foxn1-independent

Keratin+Medullary TEC

Cortical TEC

Origins

Figure 1Major cell types in the thymus and their developmental origin. The thymus can be divided intohematopoietic cells (CD45+) that are transient passengers and resident stromal cells (CD45). CD45cells include two lineages: Thymus epithelial cells (TEC, Keratin+) that originate from pharyngealpouch endoderm (third pouch in the mouse) and mesenchymal cells (Keratin), which are a mixture ofcell types that contribute to various structures of the thymus such as capsule or vasculature. The origin ofthe mesenchyme appears heterogeneous. The ratio of CD45+ to CD45 cells is about 50 to 1, but mostof the depicted cell types can be isolated based on phenotype from thymus cell suspensions followingenzymatic digestion of the thymus.

phenotypes are not easily distinguishable be-cause defects in one cell type may cause alter-ations in other cell types as well. This limi-tation also holds true for conclusions drawnabout the relationship between overall organarchitecture and T cell development, and viceversa, or, in short, the concept of crosstalk(21).

Until about 2001, the prevailing view ofthymus organogenesis was, at least in part,based on early morphological studies that hadto rely on extrapolation from standing pic-tures to moving cells (22). Anatomical butalso functional data were obtained by graft-ing experiments in developing birds (23, 24).Collectively, these studies led to the view that

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epithelial cells of endodermal and ectodermalorigin (22) and mesenchymal cells of neu-roectodermal (neural crest) origin (23, 24)all contributed directly or indirectly to thethymus. Directly means that cells and theirprogeny constitute thymus structure, as is thecase for pharyngeal pouchderived epithe-lium, and indirectly means that cells provideinductive signals in trans. Neural crest (NC)-derived mesenchyme is thought to fall in thislatter category. To what extent it also con-tributes to thymus structure has not been fullyresolved.

The mechanism that establishes the sepa-ration into the inner, morphologically lighterzone, the medulla, and the outer, morpholog-ically darker zone, the cortex, had apparentlybeen settled many years ago. A look at the evo-lution of the immune system strongly suggeststhat medulla-cortex organization is function-ally important because, as soon as there wasa thymus, this architectural hallmark of thethymus was present (25). According to an ear-lier model of cortex and medulla development(22), a layer of pharyngeal pouch epithelium(endoderm) was the source of medullary ep-ithelium, whereas its surrounding cortex wasderived from a layer of ectodermal epithelium.The latter was donated by the cervical vesicle,ectodermal epithelium that comes closely intothe proximity of the endodermal pouch at onepoint in ontogeny (embryonic day 10.5 in themouse). Hence, medulla-cortex organizationwas supposed to be established from two celllayers by a mechanism involving invaginationand circumferential growth. The model im-plying cell layer movements was revised by thending that the medullary epithelium is com-posed of single epithelial cellderived isletsthat coalesce to form larger medullary areas inthe adult thymus (13, 26). Hence, the medulladevelops from few progenitors, and the ex-tent of progenitor proliferation establishes theboundaries between medulla and cortex.

The remarkable capacity of cell suspen-sions of puried fetal thymus epithelial cellsto reaggregate in vitro (11) and to form a func-tional thymus when grafted under the kid-

ney capsule (12) paved the way to examinethe potential of fetal epithelial cells, or sub-sets thereof. Based on this approach, a TECstem/progenitor phenotype was proposed afew years ago (14, 15), but the exclusivity ofthis phenotype was questioned recently (27).Hence, the search to identify, enumerate, andfunctionally characterize true thymic stem orprogenitor cells is only beginning. In fact, itis not clear whether self-renewing thymic ep-ithelial stem cells exist, and if so, to what ex-tent they are involved in the generation, orregeneration, of the thymus.

Grafting techniques were further renedby mixing in single, genetically marked cellsinto a donor thymus, followed by graftingof this tagged tissue, and subsequent visual-ization of single cellderived medullary andcortical TEC progeny (26, 28, 29). More-over, genetic activation of single thymic ep-ithelial cells in athymic nude mice showedthat one TEC progenitor could form small yetfunctional thymus units, again composed ofmedulla and cortex. This can happen, at leastexperimentally, early after birth in the thoraxand hence later than normal and away from itsnormal physiological place, the third pouch(26; discussed in 29). On the basis of dye-marking and embryo culture techniques andin line with earlier studies on thymus devel-opment in birds (23), researchers abandonedthe dual germ layer origin model of mousethymus epithelium in favor of a single endo-dermal origin model (30, 31). By denition,the identication of clonal common medullaand cortex progenitors also calls for a commongerm layer origin (26, 28).

The structural components of the thy-mus have been only poorly accessible for along time, mostly owing to difculties or in-efciency in retrieving these cells from thesolid organ. This, however, is a prerequisite,for instance, to probe the stromal cell sub-sets functionally (10) or phenotypically (8).Monoclonal antibodies and other reagentsthat specically recognize subtypes of stro-mal cells have been instrumental in dissectingthe thymus structure (8, 3236). Mutations

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can now be introduced into thymus epithe-lium by tissue-specic gene targeting (37) thathas been applied to thymus stromal cells (38,39), or by nude mouse [ forkhead box N1 gene(Foxn1nu/nu)] blastocyst complementation (9).Mice expressing Cre recombinase specicallyin ancestors and/or their progeny of thymusepithelial cells (Foxn1Cre) (39, 40) will proveuseful not only for studying gene function inthymus organogenesis but also for clarifyingthe origin and lineage relationship of stro-mal cell subsets (fate mapping). Strategies forgene targeting in, and fate mapping of, thy-mus epithelial cells are depicted in Figure 2.Visualization of thymus epithelium by expres-sion of markers such as enhanced green u-orescent protein (Egfp) (Foxn1Egfp) (41) orenzymes (Foxn1LacZ) (39, 40) under the con-trol of TEC-specic genes should also shednew light onto the development and mainte-nance of TEC and reveal temporal and spe-cial changes in expression patterns of TECgenes. Finally, genetic screens in vertebratesother than mice, e.g., zebrash, are ongoingand aim at the identication of new genes thatcontrol thymus organogenesis (42, 43).

Rather than attempting a comprehensivecoverage of all current knowledge of thymusorganogenesis, this review focuses on signi-cant recent advances in the eld, starting witha brief primer on the embryological origin ofthe thymus in phylogeny and concluding withthe recently identied functional second thy-mus in mice, located in the neck.

PHYLOGENY OF THYMUSORGANOGENESIS

The appearance of the thymus in evolutionis linked to the appearance of lymphocytesexpressing highly diverse antigen-recognitionreceptors based on DNA recombination ofvariable (V), diversity (D), and joining ( J) geneelements. This diversity, combined with strin-gent selection processes forced onto devel-oping lymphocytes, allowed for self-nonselfdiscrimination that is a condition of cell-mediated adaptive immunity (4447). The

thymus evolved as the primary lymphoid or-gan to fulll these functions, that is, gener-ation of a large and selected T cell reper-toire. In light of recent ideas on anatomicalmicrocompartments that serve as specializedhematopoietic and stem cell niches, it is note-worthy that T cells required an entire organ,and not merely a niche. The thymus is anautonomous organ, physically separated fromthe general primary hematopoietic sites suchas the bone marrow. The destructive potentialof T cells, once released into the body follow-ing incomplete or faulty selection in an onlypoorly separated niche, could have necessi-tated the emergence of an entirely separateorgan.

No thymus is known in species more prim-itive than vertebrates. Among vertebrates,only jawed, and not jawless (agnatha such aslamprey), species have a thymus (4446). Theprecise embryological origin of the thymus,the number of thymus organs per animal,and the nal anatomical positions of thymuslobes all differ markedly in different species(Figure 3) (reviewed in 48). The commontheme is that the thymus always originatesfrom pharyngeal pouches that arise as special-ized pockets of the foregut endodermal tube.Pharyngeal pouches harbor primordia for or-gans and tissues later found in chest, neck, orhead regions, including the thymus, and theparathyroid gland (49). The origin of the thy-mus in the inner layer of an embryonic gut an-cestor is reminiscent of GALT (gut-associatedlymphoid tissue), which is a key lymphoidstructure in species prior to the appearance ofa thymus. Thus, the thymus may have evolvedas a GALT derivative (45). The most primitivethymus-bearing species are cartilaginous sh(e.g., sharks and rays). In sharks, thymus anla-gen are located in the second to sixth pouch,whereas they are found in the second pouchin frogs, in the second and third in reptiles,and in the third and/or fourth in bony sh,birds, and mammals (Figure 3). Thus, speciesare exible in positioning of the thymus an-lage somewhere along the pharyngeal foregutendoderm. Numbers and positions of the


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nal thymus, or thymi, can also be variable.Chickens have seven, sharks ve, and urodele(e.g., salamander) amphibians three thymuspairs, while many teleost sh species, anuranamphibians (e.g., frogs), and many mammalshave only one thymus composed of two bilat-

eral lobes. The position of thymus in the neckand/or in the chest in different mammals isdiscussed in the context of the cervical thy-mus in the mouse.

In some species, each thymus has a privateanlage. For instance, in sharks, ve thymus

Nude blastocyst complementation

Strategies for gene targeting in and fate mapping of thymus epithelium

Conditional knockout or marker switch driven by Foxn1Cre, KeratinCre, or other loci

Foxn1nu/nublastocyst

Foxn1+/+ ES cellswith homozygousmutation in gene of interest

Advantage

Principle

Disadvantage Nude blastocyst complementation is laborious because it requires generatingchimeras by repeated blastocyst injections. In addition, homozygousnull ES cells are required, and the method is constitutive and notconditional.

Foxn1Cre mouse KeratinCre mouseOthers

Cre-dependent deletion of floxed genes in TEC or Cre-dependent activation of marker genes in TEC

Mouse with floxed genes of interest or floxed stopper preventing marker gene expression in the absence of Cre

X

Incomplete or late deletion via Foxn1Cre

(or KeratinCre) may lead to a mosaic in TEC.The spatial distribution of deleted versus nondeleted TEC is a challenge to analyze.

a b

Foxn1Cre drives Cre expression in all orthe vast majority of Foxn1-expressing cells at any time in ontogeny, leading to homozygousdeletion in floxed alleles in mTEC and cTEC.This also works using KeratinCre mice, orCre mice that drive expression of Cre in allor subsets of TEC.

The Foxn1 gene acts in cis in TEC.Hence Foxn1nu/nu cells cannot be rescued by Foxn1+/+ cells in trans.In Foxn1+/+ ES into Foxn1nu/nu

blastocyst chimeric mice, TEC originatefrom Foxn1+/+ ES cells. Hence, TECin such mice bear the homozygous mutations from the ES cells.

Nude blastocyst complementation is arguably a technique forcing most, if not all, TEC to carry the desired mutation.

Tissue-specific gene targeting of TEC is a versatile approach that could be applied to all floxed genes of interest.

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primordia each give rise to one thymus lobepositioned along each side of the body. Thus,multiple thymi arise in a one-anlage-to-one-thymus ratio (Figure 3). In contrast, in thechicken, the anlagen in the third and fourthpouches give rise to one immature thymusthat subdivides secondarily into multiple in-dividual thymus lobes positioned along theneck (Figure 3) (50). As we begin to think interms of organ progenitor cells, it is likely thatthe original pharyngeal anlage in the chickenharbors a certain number of progenitor TECthat are partitioned into separate cell clusters,each of which gives rise to one nal thymuslobe.

CELLULAR BASIS OF THYMUSORGANOGENESIS:ENDODERMAL EPITHELIUMAND THE GERM LAYER ORIGINOF THE THYMUS

In analogy to general organ development, thy-mus organogenesis has been divided into sev-eral consecutive steps: (a) positioning; (b) bud-ding and outgrowth of the thymus anlage fromthe third pouch; (c) detachment of the prim-itive thymus from its endodermal basis; and(d ) patterning, differentiation, and migrationof the thymus toward its nal anatomical posi-tion (51, 52). Positioning refers to pouch for-mation at the prospective site where epithelialcells will later undergo commitment toward

thymus and parathyroid fates. Morphologicalthree-dimensional reconstructions (22) indi-cate that, on embryonic day 9 (developmentaltiming is from analyses of the mouse), the pha-ryngeal pouch constitutes a double-layeredmembrane composed of an ectodermal and anendodermal cell sheet. On day 9.5, these layersblend together, and it is likely that the precisegerm layer origin of these epithelial cells inthe third pouch can no longer be assigned withcertainty solely based on morphology. Later,on day 10.5, parts of the ectodermal cervicalvesicle come into close contact with the en-doderm of the third pouch. It was thoughtthat these ectodermal cells rapidly proliferateand nally surround the endodermal tissue.These and earlier (53) observations formedthe basis for the long-held textbook view (5456) of a double germ layer origin of the thy-mus with endodermal and ectodermal originsof medullary and cortical epithelium, respec-tively. In contrast, on the basis of grafting ex-periments in birds, researchers concluded thatthymus epithelium is derived from a singlegerm layer, the endoderm (23, 24), that re-quired the presence of NC mesenchyme (seebelow).

More recently, the question of the germlayer origin of thymus epithelium was read-dressed, this time in the mouse (30). Examina-tion of the histiogenesis of the thymus duringthe critical period (E10.5 to E12) conrmedthat third pouch endoderm and third cleft

Figure 2Strategies for gene targeting and fate mapping of thymus epithelium. (a) Mutations can be targeted to thethymus epithelium, and gene function can be analyzed in TEC by the generation of chimeric mice madeby injection of Foxn1+/+ embryonic stem (ES) cells into nude (Foxn1nu/nu) blastocysts, termed nudeblastocyst complementation (9). This strategy takes advantage of the fact that all thymic epithelial cells insuch chimeras (9), as well as in aggregation chimeras of Foxn1+/+ and Foxn1nu/nu embryos (149, 150), arederived from the Foxn1+ origin because the nude gene acts in cis and cannot be rescued in trans (149).The use of Foxn1+/+ ES cells bearing homozygous null alleles in a gene of interest will result in a thymusin which TEC lack the gene that is deleted in the ES cell. Nude complementation has been used to studythe role of vascular endothelial growth factor (Vegf )-A in thymus epithelium, a gene that is heterozygouslethal if mutated in all cells of an embryo. (b) Thymus epithelium can be genetically modied usingTEC-specic Cre recombinase deleter mice such as Foxn1Cre (40), or various KeratinCre (e.g., 26) mice.These lines can be used to delete oxed genes or to activate Cre-dependent reporter loci (fate mapping).The delity and inclusiveness of the Cre activity determines how specic and how quantitative theexperiment will be. Advantages and disadvantages are listed.


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imyhTsehcuop laegnyrahP

Shark

a

b

c

Chicken

pp1

pp2

pp3

pp4

pp5

pp6

pp3

pp4

Mouse Cervical thymus

Thoracic thymus

pp1

pp2

?

pp1

pp2

pp3

pp4

pp5

pp6

Figure 3Origin of the thymus in phylogeny: a variation of the theme. Aphylogenetic comparison of the origins of the thymus in shark (a),chicken (b), and mouse (c) demonstrates that thymus anlagen can be foundin different pharyngeal pouches (pp) and that the ratio of mature thymuslobes per pharyngeal pouches can differ. In the shark, each thymus lobehas its own anlage (a), whereas the chicken splits one thymus, from twopouches, into seven lobes (b). In the mouse, the thoracic thymusoriginates from the third pouch. It remains to be determined whether thecervical thymus in mice develops akin to the chicken or the shark thymus.

ectoderm indeed make contact. Evidently, thiscontact does not result in a compound struc-ture incorporating cells of both germ layerorigins. Instead, there are signs of apoptosisat the contact site, suggesting a cell loss, pre-sumably on the ectodermal side (30). More-over, when the outer pharyngeal surface ofE10.5 embryos was dye-labeled in vitro andthese ectodermally tagged embryos were cul-tured for 30 h, there were no labeled cellsfound in the thymus. This indicates, again,that ectodermal epithelium does not con-tribute to the thymus proper. In addition tothese experiments involving in vitro dye la-beling and embryo culture techniques (57),endoderm-only third pouch tissue was dis-sected from E9 embryos and grafted into nuderecipients. These grafts developed into func-tional thymi with medulla-cortex architecture(30).

There is also genetic evidence for acommon germ layer origin of corticaland medullary epithelium from tetraparentalchimeric mice generated by injection of EScells into MHC-mismatched blastocysts (13).There was no clear relationship between theorigin of mTEC islets, discussed later in thecontext of TEC progenitors, and the ori-gin of the cortex immediately surroundinga medullary islet (13). In other words, amedullary islet of ES cell origin could be lo-calized in cortical epithelium of its own (ES),but also of the opposite (blastocyst) origin.Hence, there was no sign that a common ori-gin translates into local units composed of ad-jacent mTEC and cTEC. However, a com-mon germ layer origin of mTEC and cTEC,and a late developmental split into mTEC andcTEC, might imply that, overall, the originsof mTEC and cTEC are proportional. Therelative contribution of the ES cell and theblastocyst to the tested organs (thymus, skin,liver, heart) was random, suggesting that theseorgans developed independently (Figure 4).In contrast, comparison of the overall contri-bution of ES cell or blastocyst to medullaryand cortical epithelium revealed a straightline pointing at a common origin and close

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relationship of cells forming medulla and cor-tex in ontogeny (58). These data also im-ply that the common origin holds true forthe entire thymus epithelium. Collectively,independent experimental approaches havenow provided compelling evidence that thy-mus epithelium is derived from the endoder-mal layer of the third pharyngeal pouch only(reviewed in 52, 59).

Budding and outgrowth of the thymus an-lage from the third pouch coincides with onsetof expression of the Foxn1 gene (60). Foxn1 ex-pression is rst detected in a subset of epithe-lial cells in the third pouch on embryonic day11.5 in mouse development (6163). Double-staining for expression of Foxn1 versus glialcells missing 2 [Gcm2], a transcription factorgene required for parathyroid organogenesis(64), reveals adjacently located epithelial cellclusters whereby Foxn1+ cells are located inthe ventral part of the third pouch while Gcm2marks the dorsal aspects of the same pouch(62). Because Foxn1 and Gcm2 are essential forthymus (60, 61) and parathyroid (64) devel-opment, respectively, these cell clusters likelyrepresent the anlagen for each or the two thirdpouchderived organs. However, a direct andexclusive precursor-product relationship be-tween Foxn1- or Gcm2-expressing cells in thepouch and the mature organs has not beenestablished. This would require the purica-tion of cells based on their expression of thesetranscription factors and a prospective test oftheir potential.

CONTRIBUTION OF NEURALCREST (NC) MESENCHYME TOTHYMUS ORGANOGENESIS:TRANSIENT OR LONGLASTING?

NC-derived mesenchyme is crucial for thy-mus organogenesis and thymus function (re-viewed and further references in 1, 51, 58,65, 66). In the original description of thisphenomenon in bird embryos, it was demon-strated that NC-derived mesenchyme col-onizes the branchial arches, surrounds the

Blastocyst origin (%)ES cell origin (%)

a b

c d

e f

Ski

n

Thymus

Hea

rt

Thymus

Liv

er

Thymus

Co

rtex

Medulla

Liv

er

Skin

Hea

rt

Skin

100

100

50

50

0

0

Figure 4Common origin of medullary and cortical TEC in tetraparental (chimeric)mice. Mice generated as described by injecting embryonic stem (ES) cellsinto MHC-mismatched blastocysts (13) were analyzed for ES cell versusblastocyst contributions to skin, heart, liver, and thymus utilizingmicrosatellite differences in ES cell and blastocyst genomic DNA. Thecontributions of ES cell and blastocyst to thymus medulla and cortex weredetermined by histological analysis of the MHC class II haplotypesindicative of ES cell or blastocyst origin. None of the organs showed anylinkage of the origins except for thymus cortex and medulla.


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thymic epithelium, and forms perivascularmesenchyme (23). Ablation of cephalic NCin birds prevented any contribution to or in-duction of thymus epithelium by NC-derivedmesenchyme, and this resulted in small thymiwith delayed development and poor function(24). These early reports were conrmed inprinciple and extended by many subsequentexperiments that showed an important rolefor NC in thymus development in vivo (re-viewed in 51, 52) and in organ culture sys-tems (10, 67, 68; reviewed in 1). Examplesof genetic pathways involved in mesenchyme-thymus epithelium interactions are providedbelow.

Given that mesenchymal-epithelial con-tact takes place early in development and thatmesenchymal cells are abundant in the adultthymus, as specied in the introduction, it hasbeen debated whether or not embryonic NC-derived mesenchyme and adult thymus mes-enchyme share a precursor-product relation-ship (reviewed in 66). Several reports addressthis point in mice generated with the aim toexpress Cre recombinase specically in theNC lineage. In combination with appropriateCre activitydependent reporter mice, NC-derived cells and their progeny should be per-manently labeled in this system. Mesenchymemarked by Wnt1Cre surrounds the E13.5 thy-mus as a massively stained, thick layer. How-ever, labeled cells become rare as soon asthe thymus grows, owing to the rapid prolif-eration of thymocytes. In the adult thymus,the overall contribution of Wnt1Cre-markedcells appears too low to account for majormesenchymal components such as the cap-sule, the septae, or intrathymic broblast (66,69). Some cells of unknown character are,however, present in the adult thymus (66). Asimilar result of transient but not permanentparticipation was obtained using a differentNC-specic marker gene, myelin protein zero(P0). By ow cytometry on thymus cells fromE13.5, as many as 30% of all stromal cells arelabeled by P0Cre (70), which is the rst quan-titative estimate of the contribution of NC tothe developing thymus. Again, few marked

cells persist at later stages. In vitro, P0Cre-marked cells from the fetal thymus could bedeveloped into melanocyte and glial cell lin-eages, further supporting their NC origin.The idea that the thymus contains cells relatedto neuronal/glial lineages is not new (71), butthere is currently no evidence to dene func-tions for such cells in the thymus. Finally, acautionary note may be warranted here be-cause, as in other fate mapping experiments,the result will ultimately depend on the strat-egy used. Random transgenic integration mayyield more mouse-to-mouse variability thantargeted knockin approaches, and the result,here low or no contribution of NC cells tothe adult thymus, depends on the propertiesof any particular Cre driver mouse and thesensitivity of the reporter detection.

A possible molecular link between NC-derived mesenchyme and thymus epithe-lium is provided via broblast growth fac-tors (Fgfs, also termed keratinocyte growthfactor [KGF]) and their receptors (FgfR).Fgf7 and Fgf10 are expressed by the mes-enchyme surrounding the embryonic thymusepithelium, and the latter expresses FgfR2-IIIb. Defects in this signaling pathway per-turb thymus development (72), demonstrat-ing a growth-promoting role for mesenchymetoward thymic epithelium. Signals via Fgfsalso induce TEC proliferation (73, 74) andprotect thymus epithelium from injury byirradiation (75) or by conditions of graft-versus-host disease (76). Another role of thy-mus mesenchyme could be the presentationof growth factors such as IL-7 or c-kit ligandto thymocytes, but currently no data supportthis idea.

GENES AFFECTING THYMUSDEVELOPMENT PRIOR TOTHYMUS SPECIFICATION

Mutations in a number of genes, includingHoxa3 (77), Eya1 (78, 79), Six1 (79, 80), Pax1(8184), Pax3 (85), Pax9 (86, 87), and Tbx1(88), lead to thymus aplasia, or hypoplasia,or failure of the thymus lobes to migrate

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toward the chest (reviewed and further ref-erences in 51, 52, 89). These genes are ex-pressed in multiple cell lineages during de-velopment, and hence their loss of functioncauses pleiotropic defects in embryonic devel-opment. It is, therefore, often difcult to dis-tinguish the primary function of these genesin thymus organogenesis, e.g., in TEC pro-genitors, from an upstream function, e.g., information or patterning of pharyngeal struc-tures or in NC migration. It is obvious thatthe thymus cannot develop normally if thethird pouch is absent as a scaffold in whichTEC progenitors arise. The effects of suchmutations are therefore upstream of thymusorganogenesis itself. Moreover, it is also pos-sible that some of these genes are requiredboth upstream of organogenesis and later inthe thymus epithelium itself. Along this line,Hoxa3 (90), Pax1 (82), and Pax9 (91) are ex-pressed in thymus epithelial cells. Because thiswas mostly measured by RT-PCR in cell pop-ulations, frequencies and phenotypes of TECexpressing these genes are unknown. TEC-specic deletion of such ubiquitous pathwaygenes will be required to resolve their functionin thymus development beyond the generaldefects that may be nonspecic for the thy-mus. An example of TEC targeting was block-ing of Bmp signaling in TEC by expression ofNoggin under the control of the Foxn1 pro-moter. Whereas blocking of Bmp signaling inpremigratory NC by transgenic expression ofNoggin in NC affected thymus developmentindirectly (92), Foxn1-driven inhibition of theBmp pathway demonstrated, in addition tothe NC defect, a role for Bmp signaling in-trinsic in TEC (93). This is compatible withexpression of Bmp family members in the thy-mus epithelial anlage (94).

Another interesting gene that belongs tothis gene category, and that is related toDiGeorge syndrome (9598), is the T boxgene Tbx1. DiGeorge syndrome is causedgenetically by heterozygous deletions withinchromosome 22q11 and clinically by malfor-mations of pharyngeal arch arteries (cardiac

outow tract) and heart, parathyroid hypopla-sia, and absence or ectopic location of the thy-mus (96, 99). Hallmarks of this phenotype arerecapitulated in mice lacking Tbx1 (88, 100),a gene that is located in the deleted regionin humans (88, 100). Tbx1/ mice displayagenesis of pharyngeal pouches 24 and con-comitant loss, or malformation, of pharyn-geal pouchderived organs and tissues (thy-mus, parathyroid gland, cardiac outow tract)(88, 100). Tbx1 is expressed in the pharyngealpouch endoderm but also in the core meso-derm of the pharyngeal apparatus and thepharyngeal ectoderm but not in NC-derivedmesenchyme (101). Thus, Tbx1 expressionmay play distinct roles in different anatom-ical sites (e.g., endoderm versus mesoderm)during development. Pharyngeal pouches failto develop in mice in which the Tbx1 de-ciency is restricted to the endoderm by meansof preferential deletion in pharyngeal endo-derm using another Fox family gene locus ex-pressing Cre, Foxg1Cre. Hence, this mutantrecapitulates the defects known from the con-stitutive null mice, including absence of thethymus (102). This demonstrates that Tbx1expression in the pharyngeal endoderm is re-quired for thymus development. However,mice that lack Tbx1 expression selectively inthe pharyngeal mesoderm, but not the endo-derm, also have a hypoplastic pharynx withimpaired pharyngeal endoderm and lack athymus (103). Conditional reversion from adefective to a functional Tbx1 allele in pha-ryngeal mesoderm, but not endoderm, is suf-cient to rescue major defects known from theTbx1 null phenotype (pharyngeal patterning,cardiovascular defects) but does not restorethymus development (103). In conclusion, ex-pression of Tbx1 both in the pharyngeal coremesoderm and in the pharyngeal endodermis a prerequisite for thymus development.Within the third pouch, Tbx1 expression co-incides rather with the parathyroid than thethymus anlage (104), and it remains to be de-termined if Tbx1 is expressed in TEC andwhether it plays a role in their development.


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EPITHELIAL PATTERNINGAND DIFFERENTIATION,AND CROSSTALK BETWEENTHYMOCYTESAND EPITHELIUM

Coinciding with hematopoietic colonizationthat initially occurs around E12 and priorto vascularization, the immature thymus un-dergoes further patterning and differentiation(reviewed in 51, 105, 106). Morphologically,this stage leads to the rst signs of medulla-cortex separation. This compartmentalizationis associated with changes in keratin expres-sion patterns in the epithelium. During on-togeny, and in the adult thymus, TEC sub-sets express different members of the keratinfamily. Major populations of adult mTEC andcTEC have been distinguished by their ker-atin (K) 5+K8 and K5K8+ phenotypes, re-spectively (107, 108 and references therein).This dichotomy is, however, not absolute be-cause K5 is also expressed in some cTEC, andK8 is also found in some mTEC (107). Re-garding the onset and pattern of K5 and K8expression in ontogeny, third pouch epithe-lium at E11.5 was reported as K5K8+ (15,108), whereas others found coexpression ofK5 and K8 already at this stage, even withhigh expression of K5 (14). A prominent TECpopulation coexpresses K5 and K8 on days 12and 13 (14, 15, 108). Using these and othermarkers, so-called double-positive TEC arethought of as progenitors of mature single-positive K5+K8 medullary and K5K8+ cor-tical TEC phenotypes (108110). This ideawas based on the fact that K5+K8+ cells pre-cede the appearance of mature K5+K8 andK5K8+ TEC in ontogeny. Moreover, largeclusters of K5+K8+ TEC are maintained inmutants with massive early blockade in T celldevelopment such as compound KitW /W andcommon chain ( c) (111), or RAG2/ c

mice (thymus stromal phenotypes reviewedin 105). Thirdly, and further discussed in thecontext of TEC progenitors, MTS24+ TEC,a controversial TEC progenitor phenotype(27), also coexpresses K5 and K8 (14, 15). In

any case, the initial patterning of the embry-onic thymus is dependent on expression ofFoxn1 in the epithelium but is independentof hematopoietic colonization.

The transition from immature TEC phe-notypes, such as abundant K5+K8+ cells, tothe full medulla-cortex organization is per-turbed in mice in which T cell development isblocked at immature stages. That the TECarchitecture is somehow inuenced by thepresence or absence of particular stages ofthymocytes development has been viewed asan interdependence between thymocytes andstroma (36, 107, 112) and has been referredto as crosstalk (21). In the original case, it wassuggested that the thymus stroma was perma-nently damaged unless it had proper contactwith developing pro-T cells (CD44+CD25)and that this contact needed to take placeat fetal stages of TEC development (112).The transgenic mouse (hCD326tg) on whichthese conclusions were based expressed 4060 copies of a human CD3 transgene (113,114). T cell development in this mouse wasblocked at an early CD4CD8 (double-negative) stage prior to expression of CD25,and numbers of residual thymocytes were verylow. This paucity of thymocytes and the se-vere block in thymocyte development weresuspected as the cause of aberrant adult TECstructure characterized by poorly discernablecortex and an abundance of cells coexpress-ing K5 and K8 (107, 112). Subsequent studiesfound, however, that the hCD326tg thymuswas not simply devoid of thymocytes, but har-bored B cells (114). Hence, it cannot be ex-cluded that it is not the absence of developingthymocytes that perturbs thymus organogen-esis but the aberrant B cells or their develop-ment that contributes to the adult stromal cellphenotype. This interpretation would ques-tion the specicity of crosstalk between pro-Tcells and TEC.

Another concern regarding models ofcrosstalk in thymus organogenesis has beenthe inability to distinguish alterations in theepithelial compartment owing to lack of

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signals from developing thymocytes to TECfrom epithelial reactions to a state of inactiv-ity. The latter scenario could lead indirectlyto defects in the maintenance of the epithe-lial cells. Along this line, a recent reevalua-tion of the developing thymus in hCD326tgmice found normal proportions of immatureK5+K8+, as well as single-positive K5+K8

and K5K8+ TEC when compared to wild-type thymus (115). These data imply that thy-mus organogenesis may be quite normal inhCD326tg mice and that the architectural al-terations seen in this mutant are secondary toregular TEC development, a conclusion thatwould support the argument against a role ofpro-T cellmediated signaling for the devel-opment of thymic epithelial cells (115). Thesuggestion that the thymus stromal cell archi-tecture is damaged at long-term unless pro-T cells contact the epithelium at the propertime in ontogeny is also contradicted by ex-periments made in KitW /W c mice, a mutantin which thymocyte development was com-pletely abrogated (116). The severely dysmor-phic thymus structure of this mutant couldbe reverted to a structurally normal and func-tional thymus when grafted postnatally into arecipient that provided wild-type hematopoi-etic stem cells (111). Finally, the fact that thehuman thymus of severe combined immuno-deciency (SCID) patients can be reconsti-tuted by transplantation of normal bone mar-row stem cells (117) further supports theargument against continuous stroma defectscaused by a block in T cell development.

Collectively, much speculation has sur-rounded the interesting crosstalk concept. Acrosstalk mechanism should involve cell sur-face molecules such as receptor-ligand pairsthat can transmit signals in both directions,and the absence of such signals might causea specic phenotype on the nonreceivingend. Some molecularly dened cases for bidi-rectional crosstalk between thymocytes andthymus epithelium exist. One documentedexample of a crosstalk mechanism fromthymocytes into epithelial cells is the defect

in the mTEC compartment in mice lackingcomponents of the TNF-TNF receptorfamily. Expression of the lymphotoxin-receptor (LTR) on thymocytes and of aLTR ligand on mTEC are required fornormal cellularity and architecture of mTEC.Mutations that interrupt this signaling path-way lead to structural defects associated withfaulty selection and autoimmunity (118).Moreover, the recent nding that expressionof RANK ligand on a CD4+CD3 inducercell population promotes the maturationof RANK-expressing CD80Aire mTECprogenitors into CD80+Aire+ mTECs (119)can also be viewed as a form of crosstalk,albeit between a highly specialized and rarelymphoid cell and a specic stage of TEC.

There are other candidate molecules thatcould mediate signaling from stroma to thy-mocytes and back. For instance, the recep-tor tyrosine kinase Kit is expressed on pro-and pre-T cells, and the membrane-boundligand [Kit ligand (KL), or stem cell factor(Scf )] is expressed on stromal cells (120). Kitsignaling into thymocytes via expression ofKL in TEC is crucial for T cell develop-ment (121), and KL can indeed signal intoepithelial cells (122). However, T cell devel-opment was permissive in a KL mutant (Sl17H )(H.-R. Rodewald, unpublished data) in whichthe cytoplasmic tail of KL is nonfunctional(123). Hence, there is currently no evidencefor KL-mediated crosstalk in the thymus. Fur-ther potential examples are Notch and Notchligand pairs. Notch-1 is expressed on pro-and pre-T cells, and Notch ligands, includ-ing Delta-like (Dll)1 and Dll4, are expressedin thymus epithelium (reviewed in 124, 125).Dll1 or Dll4 could signal into the epitheliumand induce changes in the microenvironmentthat could ultimately be involved in the thy-mus phenotype of Notch-1 mutants, includ-ing an abundance of B cells instead of T cellsin the thymus (124, 125). However, it is notknown whether Notch-Notch liganddrivenmechanisms play a role in crosstalk or in TECdevelopment, as has been speculated (28).


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POTENTIAL, FREQUENCIES,AND PHENOTYPES OFTHYMUS EPITHELIALSTEM/PROGENITOR CELLS

Progenitors for mTEC

Measurements of stem or progenitor cell ac-tivity, and the prospective isolation by phe-notype and subsequent analysis of progenitorpotential, ultimately require clonal assays. Al-though single cellbased assays to study TECdevelopment in vitro are still lacking, cellularand genetic strategies have been developed tofollow the fate of single TEC progenitors ortheir activity in vivo (13, 26, 28). Clonal pro-genitor activity for TEC was initially iden-tied for the medullary lineage in chimericmice and in grafts of reaggregate fetal thy-mus organ cultures (RFTOC) (13) and has re-cently been conrmed independently by ge-netic means (26). The former approach wasbased on techniques that allow disassembly ofembryonic or fetal thymus by enzymatic di-gestion, purication of stromal cells or subsetsthereof, and reassembly into thymus reaggre-gates in vitro (10). These RFTOC are func-tional in that they support T cell developmentin vitro, but the TEC architecture of RFTOCin vitro is quite different from that of a normalthymus in vivo.

In RFTOC assembled from puried TECin the absence of thymocytes, distinct mTECor cTEC phenotypes were present, but thesecells did not form medulla-cortex structures.In contrast, RFTOC grafted into recipientmice not only supported steady-state T celldevelopment, but also achieved proper thy-mus architecture, including regular medulla-cortex organization (12, 126). This remark-able capacity of TEC to build functionalthymus architecture from reaggregated cellsuspensions suggested that mTEC and cTEC,randomly arranged in the reaggregates invitro, could migrate within the graft to seg-regate into cortex and medulla. Such sortingout would have required active migration, orat least directed movement, and recognition

of the neighboring cells as mTEC or cTEC.However, experiments using RFTOC assem-bled from two distinct donor strains, followedby transplantation of these mixed RFTOC,revealed an alternative mechanism and pro-vided evidence for clonal events during ep-ithelial organogenesis of the thymus (13; re-viewed in 106). Cellular products of mTECprogenitor activity were visible in the formof epithelial medullary islets, the majority ofwhich were either of one or the other, butnot of mixed, origin (13). Medullary epithe-lial islet formation also occurred during nor-mal thymus organogenesis in vivo, as shownin chimeric mice made by injection of ES cellsinto MHC-mismatched blastocysts. Here, in-dividual epithelial islets stemmed from eitherES cell or blastocyst origin. Hence, medullaryislets arise from single progenitors. With age,the islet-like character of mTEC is harder torecognize, as many mTEC islets coalesce toform conuent regions of medullary epithe-lium. The existence of clonal medullary isletswas recently conrmed genetically (26) (seebelow), and the principle of islet formationin chimeric thymus grafts has been used asan assay to assess the potential of phenotypi-cally dened embryonic mTEC subsets (127)or the role of MHC class II expression forthe selection of Foxp3+ regulatory T cells onmedullary epithelium (128).

Serial sectioning showed that one thymuslobe of a mouse at two weeks postnatal con-tained 300 medullary areas (according tothis denition, a medullary area is larger thana medullary islet). These medullary areas werecomposed of one to three islets. Hence, upto 900 mTEC progenitors are sufcientto generate the entire medulla in one thy-mus lobe of a mouse (13). This estimate isin a strikingly similar order of magnitude asthe estimated 1500 TEC in the E13.5 thy-mus that express the tight junction proteinsclaudin (Cld)-3 and 4 (Cld3,4) (127). Alreadyon E10.5 in development, Cld3,4+ cells werefound in the apical epithelial layer in the thirdpouch thymus anlage. Subsequently, Cld3,4+

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cells showed a clustered arrangement that par-tially overlapped with general mTEC mark-ers such as MTS10 or UEA-1. In the adultthymus, Cld3,4 expression marked a subsetof mTEC expressing the autoimmune regu-lator gene Aire (127). Cld3,4+Aire+ cells arepresumably fully mature mTEC that are busyin promiscuous expression of tissue-restrictedself-antigens (TRA) (reviewed in 5). By owcytometry, several TEC subpopulations wereidentied and separated based on Cld3,4+

and UEA-1 expression. Interestingly, Cld3,4+

cells, irrespective of UEA-1 expression, gaverise to only mTEC and not cTEC when mixedinto RFTOC and grafted into nude mice,whereas Cld3,4lowUEA-1 cells gave rise toboth mTEC and cTEC. These data suggestthat commitment to the mTEC lineage cantake place already by E13.5, at least in cellsdened by Cld3,4 expression. Collectively,the very early and restricted expression ofthe tight junction proteins of Cld3,4 marksan mTEC pathway. Because the medullaryclusters that arise from Cld3,4+ progenitorsare very similar in size and frequency to themedullary islets described earlier, it is possi-ble that Cld3,4 expression marks the entiremTEC pathway at some point in the differ-entiation tree of TEC.

It is not clear whether mTEC progenitorsare only active during ontogeny, or whetherthey continue to generate mTEC de novo inthe adult thymus. Clonogenic, mTEC-islets-forming mTEC progenitors, or their activity,have not been detected in the adult thymus.For a long time, TEC were considered post-mitotic cells that constitute an epithelial net-work that is constructed in ontogeny and latermaintained. However, several reports recentlyfound TEC proliferation indicating rapid ep-ithelial turnover in the steady-state thymus (5,20, 129). There are considerable differences inthe reported proliferation rates, ranging fromas many as 23% of MHC class II+CD45

TEC (the majority of which are mTEC) in-corporating BrdU after three days of contin-uous labeling (20) to as few as 8% of mTECincorporating BrdU after one week of label-

ing (129). This would translate into half-livesof about six days in one case versus four weeksin the other case; the latter is similar to a thirdestimate of six weeks (5).

It is obvious from the above considerationsthat there are large gaps in our understandingof the sequence of events during mTECdifferentiation from mTEC progenitors tomature mTEC. Specically, the relationshipof mTEC differentiation stages to mTECfunction (TRA expression and presentation;full maturation as antigen-presenting cells)and mTEC turnover (proliferation versus celldeath) are only poorly understood. On theone hand, islets of mTEC vary in diameterfrom a minimum of 60 40 to a maximumof 170 170 m and harbor between 5 and45 epithelial cells in a two-dimensional lattice(13). On the other hand, numbers of cellsexpressing a particular TRA in the medullaappear even lower than the cells per islet (129,130; reviewed in 5). Because an mTEC isletis originally made by a single progenitor, cellswithin an islet might be further diversiedinto subclones (131). It is not known whetherthis diversication is stable or whetherindividual mTEC change their expressedTRA pattern over time. It will be necessary tobetter dene stages of mTEC differentiationfrom mTEC progenitors via immature tomature mTEC and to shed light on the signalsthat regulate this developmental progressionas well as on the capacity of TEC at eachstage to present antigens and ultimately guideTCR repertoire selection on thymocytepopulations (5, 119, 129, 131, 132).

Common mTEC and cTECProgenitors

It was speculated early on that mTEC andcTEC share a common progenitor (109, 110).The idea was based on coexpression of mark-ers such as keratins on TEC early in thymusorganogenesis, as discussed above in the con-text of the germ layer origin. More recently,the generation of a complete and functionalthymus environment in RFTOC grafts was


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also taken as evidence of a common progen-itor for both mTEC and cTEC. Transplan-tation of RFTOC assembled from embryonicday 12.5 (14) or fetal day 15.5 (15) TEC ledto the formation of a thymus composed ofboth medulla and cortex (discussed in 133).Gill et al. (15) interpreted their data as di-rect evidence of thymic progenitor cells giv-ing rise to both cortical and medullary epithe-lial lineages, and Bennett et al. (14) arguedstrongly in the same direction. These poly-clonal (bulk) experiments in fact demonstratethat the populations of cells used to assem-ble the grafts contained all the cells requiredfor a functionally and structurally normal thy-mus. What they could not answer was whetheror not a common progenitor for mTEC andcTEC existed and, if so, whether it was re-sponsible for the generation of both mTECand cTEC in the grafts.

This key property of a common progen-itor, clonogenicity, has only recently beenaddressed (26, 28; reviewed in 29). Single ep-ithelial cells isolated from yellow uorescentprotein (YFP) expressing embryonic E12.5thymus were injected into a nonuorescenthost thymus, and such single celltaggedthymi were transplanted into recipientmice to allow for full thymus development.Immunohistological analysis showed that, ineach case of positive reconstitution, the singleembryonic epithelial cell had produced bothmTEC and cTEC. Because the donor cellswere puried via expression of a pan-TECmarker [EpCAM1; antibody G8.8 (34)], noparticular TEC subset was selected, implyingthat a large proportion of epithelial cells inthe day 12 thymus has dual potential formTEC and cTEC and that their progenypersists in the adult thymus (28).

A different approach addressing progen-itor activity in thymus organogenesis wasbased on epithelial cell tracing using geneticin situ labeling (26). Cre recombinase underthe control of the human Keratin 14 promoter(K14Cre) effectively acted as a random andvery rare switch that turned on YFP expres-sion in TEC. Although no labeled cells were

found in the thymus at birth, numbers of micewith labeled cells and numbers of YFP+ TECper thymus increased with age after birth. Atall times analyzed, labeled progeny remainedvery rare, consistent with Cre-mediated YFPexpression only in single or in very few TECprogenitors. Three patterns of progeny werenoted: (a) mTEC clusters only, reminiscentof medullary islets; (b) cTEC clusters only;or (c) mTEC plus cTEC progeny (26). Theseresults strongly suggest that K14Cre randomlymarked TEC progenitors and their progenyand that this labeling event could occur overdevelopmental stages that covered common tocommitted TEC progenitors. If the genetichit occurred at an early postnatal age, TECprogenitors, endowed with the listed poten-tialities, persist at least until that age in a nor-mal thymus. After the putative mTEC versuscTEC branch in TEC differentiation, cellsprobably migrate considerable distances. Thisis evident from the space that was observedbetween mTEC and cTEC progeny of com-mon origin in the adult medullary and corticalzones, respectively (26). The notion of TECmigration, or perhaps passive movement, dur-ing development would also t the topolog-ical dissociation of mTEC versus cTEC ofthe same origin in chimeric mice (13). Col-lectively, based on cellular (13, 28) and ge-netic (26) evidence, the thymus harbors TECprogenitors that, at the single cell level, con-tribute to the formation of thymus epithe-lial structure. Except for an estimate on to-tal numbers of mTEC progenitors required(see above), there is currently no informationon the total number of common mTEC andcTEC progenitors that normally engage inthe building of a thymus.

The marking of common TEC progeni-tors left unanswered whether or not singleTEC progenitors are capable of forming func-tional thymus units. This was addressed againin the K14Cre mouse, but in this case the Crerecombinase was used to revert a nonfunc-tional Foxn1 allele to a functional Foxn1 al-lele akin to correcting the nude mutation inthe thymus in vivo. The Foxn1nu/nu thymus is

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composed of epithelial cysts that do not sup-port T cell development. When crossed to aCre-dependent LacZ reporter, K14Cre markedrare TEC that, in this case, were located in thewall of the nude thymus cysts. When Cre re-verted the loss-of-function Foxn1 allele backto a functional Foxn1 allele, an event thatoccurred again randomly in single postnatalepithelial cell precursors, small units of thy-mus tissue developed (26). These neo-thymishowed all tested hallmarks of a normal thy-mus, including medullary and cortical orga-nization and expression of Aire. Mice bearingsuch thymi had elevated numbers of immuno-competent T cells that, unlike the few T cellsfound in nude mice, expressed a diverse TCRrepertoire. These experiments demonstratethat the block that occurs in TEC develop-ment in the nude mouse does not lead to acomplete loss of TEC progenitors. Rather,TEC progenitors may enter a stage of dor-mancy or may be continuously generated denovo in nude mice. Once genetically revertedto wild type, they can recapitulate normal on-togeny and complete their differentiation intofunctional TEC. It is noteworthy that this de-velopment can take place outside of the physi-ological location, the third pouch, because thecystic thymus rudiments in the nude mouseare at this age in the chest. For anatomicaland kinetic reasons, it is unlikely that the in-ductive signals that are provided to the thymusduring normal ontogeny, e.g., by NC-derivedcells, are available to those thymi that use theirsecond chance (29). This lack of proper con-text could at least in part be responsible fortheir small size (26).

TEC Progenitor Phenotypes

In the aforementioned single cell exper-iments, TEC progenitor cells have notbeen physically puried except by the pan-epithelial marker EpCAM1 (28). Are therecell surface markers known that identify TECprogenitors? This would be an important pre-requisite for analyses of the prospective po-tential of these cells. Previous experiments

have suggested that MTS24 is a markerfor embryonic TEC progenitors, a proposalthat met with some interest (133, 134). Asnoted before, RFTOC assembled from em-bryonic (14) or fetal (13, 15) TEC developedinto functional thymi in vivo. Although ear-lier studies showed thymus development inRFTOC grafts made from several hundredthousand fetal TEC dened as MHC classII+CD45 cells (12, 13), others reported thy-mus formation using much lower numbers ofcells using the MTS24 marker for positive pu-rication. Twelve thousand ve hundred TECfrom E12.5 (14), or 2,500 from day 15.5 fe-tal (15), contained all the cells required for athymus. Even when only 500 cells were trans-planted, nude recipients showed signs of tran-siently functional thymus (14).

In analogy to other progenitor systems, itcan be assumed that the frequency of pro-genitors is low among all TEC. Are MTS24+

TEC rare cells, as has been suggested (133)?On E10.5, MTS24 is broadly expressed in theendodermal pharyngeal pouches (15) (includ-ing those that do not give rise to a thymus).By histology, most (14, 15), if not all (27),epithelial cells on day 11.5 and 12.5 expressMTS24. By ow cytometry, essentially all thy-mus epithelial cells, gated as pan-cytokeratin+

or EpCAM1+ cells on E12.5 and 13.5, areMTS24+ (27), suggesting that at this stage ofdevelopment, MTS24 cells are nonepithe-lial cells. Consistent with these data, and notsurprisingly, only MTS24+ (that is, epithe-lial) and not MTS24 (that is, nonepithelial)cells from E12.5 have thymus-forming capac-ity (14). Is MTS24 a progenitor marker at laterstages of thymus development? On day 15.5,about half of all TEC still express MTS24before the frequency of MTS24+ cells de-clines to a few percent at later fetal and adultstages (14, 15, 27, 135). One report foundthymus potential exclusively in MTS24+ andnot MTS24 epithelial from day 15.5 thymus(15). However, a recent reassessment of TECprogenitor activity using MTS24 expression-based purication did not reproduce thesendings using larger cell numbers from


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14- or 16-day-old thymi and concluded thatboth MTS24+ and MTS24 epithelial cellswere similarly potent in forming a functionalthymus (27). The available evidence suggeststhe following order of events: MTS24 is ini-tially a nonthymus-specic marker of pharyn-geal endoderm cells; it then marks all TECaround day 12.5 and is expressed on day 15.5on a major subset of TEC. At this stage, thy-mus potential is included in both MTS24+

and MTS24 epithelial cells. From the adultthymus, MTS24+ cells can be retrieved, buttheir function remains to be determined (135).

If MTS24 is perhaps not the TEC pro-genitor marker, is there any evidence for stem-ness among TEC? Interestingly, TEC expressgenes such as Nanog, Oct4, and Sox2, which arecommonly found in stem cells, and the ex-pression of these hallmark genes is reducedin TEC from Aire-decient mice (91). Be-cause gene expression was detected by re-verse transcriptase polymerase chain reaction(RT-PCR) in populations of TEC, it will beimportant to determine the frequencies ofTEC and their maturation stages that expressNanog, Oct4, and Sox2. Perhaps TEC stem orprogenitors will be found within these cells oramong cells expressing p63 (136) (see below).

GENES AFFECTING THYMUSDEVELOPMENT AFTERTHYMUS SPECIFICATION

Foxn1, the Gene Mutatedin the Nude Mouse

There is no denitive marker that indicatesepithelial cell commitment toward thymusfate prior to expression of Foxn1. Function-ally speaking, Foxn1 is the single most impor-tant gene known to be essential specicallyfor thymus epithelial development (60, 61).Therefore, it is worthwhile to take a closerlook at properties of Foxn1 such as expres-sion, regulation, and function. Foxn1 is a pro-tein belonging to the family of forkhead boxtranscription factors (137139). In addition toFoxn1, other members of this gene family also

play important roles in the immune system,such as Foxp3, which determines the develop-ment and function of regulatory T cells, andFoxo factors, which are involved in apoptosisand proliferation, and hence leukocyte home-ostasis (138, 139). Foxn1 is characterized bya winged-helix/forkhead DNA-binding do-main and a transcriptional activation domain(42, 140). In the original nude mouse allele(Foxn1nu), a single base pair deletion in exon3 causes a frame shift leading to a truncatedFoxn1 protein lacking both the DNA-bindingand the activation domain (60). HomozygousFoxn1nu/nu mice had the same thymus and skinphenotypes as compound heterozygous micebearing one engineered null allele of Foxn1(Foxn1) and one natural mutant allele ofFoxn1 (Foxn1nu), formally showing that Foxn1is allelic to the nude gene (61).

In mice homozygous for a Foxn1 allelelacking exon 3 (Foxn1/), thymus epithe-lium developed beyond the block in Foxn1nu/nu

mice. Foxn1 encodes a Foxn1 protein with alarge deletion in the N-terminal part of Foxn1(141). The phenotype of Foxn1/ mice over-all resembled that of a hypomorphic mutant inwhich TEC phenotype and organization sug-gest an arrest at a K5+K8+ stage. In contrastto the cystic Foxn1nu/nu thymus, the Foxn1/

thymus had a more continuous structure per-missive to support T cell development, albeitonly poorly and with a delay in fetal thymocytedevelopment. It is not clear whether this TECphenotype results from a specic requirementfor the N-terminally deleted amino acids atlater stages of TEC development, perhapsbeyond the K5+K8+ stage, or whether theFoxn1 allele encodes a Foxn1 protein with anoverall reduced functional activity. The sta-bility of the Foxn1 protein encoded by thisparticular allele has not been determined.

Little is known about upstream factors thatregulate Foxn1 expression. Through trans-genic work, DNA fragments and promoter el-ements that permit gene expression under thecontrol of Foxn1 have been described (41, 93,142). It has been proposed that Foxn1 expres-sion is regulated through members of the Wnt

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family (143). Given that no single or com-pound Wnt mutant mouse has been reportedthat resembles a nude thymus phenotype, fur-ther clarication of the role of Wnt familymembers directly upstream of Foxn1 would behelpful. In brief, there is very little informa-tion on how Foxn1 transcription is initiatedin development or maintained in the adultthymus.

On the basis of a Foxn1lacZ knockin allele,in situ hybridization, and antibody staining,investigators have found that Foxn1 expres-sion in thymic epithelium is rst detectableon E11.5 (6163), a stage preceding coloniza-tion of the thymus by hematopoietic progen-itors. Analysis of a Foxn1lacZ allele demon-strates that Foxn1 is expressed in most, if notall, thymic epithelial cells both at embryonicstages and in the adult (61). However, a morerecent study that used an anti-Foxn1 anti-body suggests that both Foxn1+keratin+ andFoxn1keratin+ thymic epithelial cells existin the embryonic thymus (E13) and that thepercentage of Foxn1keratin+ TEC is even ashigh as 80% in the adult thymus (63). If sub-sets of adult TEC differ, in fact, in expressionof Foxn1, it would be interesting to dene howthose Foxn1+ and Foxn1 subsets differ withregard to the relative stages of maturation, cel-lular age, turnover, or their functional capaci-ties to promote T cell development. Althoughthe extent to which adult TEC actively expressFoxn1 is controversial, fate mapping of TECusing a Foxn1Cre allele convincingly showedthat most, if not all, TEC arise from Foxn1+

progenitors, or at least transit through a stageof ubiquitous Foxn1 expression (39, 40). Asmentioned earlier, genetic activation of Foxn1in single TEC led to the appearance of unitsof productive thymi in an otherwise nude thy-mus. This underscores the idea that Foxn1is expressed in thymus-forming progenitors(26).

Foxn1-dependent cells in the thymus canbe visualized in nude blastocyst comple-mentation (Figure 2) (9) by injection ofGfp+Foxn1+ ES cells into GfpFoxn1nu/nu

blastocysts (Figure 5). On thymus tissue sec-

a

b

c

DAPI = all nucleiGfp= Foxn1-dependent TEC

ES cellsGfp+ Foxn1+/+

Blastocysts

Gfp Foxn1nu/nu

Estimation of number and location of Foxn1-dependent TEC by visualization of Gfp+ nuclei

Figure 5Visualization of Foxn1-dependent thymus epithelium. The rarerepresentation of TEC among all thymus cells is demonstrated in chimericmice generated by injection of Gfp+ Foxn1+/+ embryonic stem (ES) cells(a) into Gfp Foxn1nu/nu blastocysts (b). Percentages, phenotypes and tissuedistribution of Gfp+ cells can be determined by ow cytometry (not shown)and by histology (c). The thymus section is from a chimera with very low(


and cTEC appear to cover different thymusvolumes per cell. The conclusion that mTECoutnumber cTEC in situ is certainly compat-ible with the observation made by many labo-ratories that proportionally more mTEC thancTEC are routinely retrieved from stromalcell preparations.

Lack of Foxn1 expression in nude micebecomes phenotypically evident as early asE12.5 or 13.5 when the Foxn1-decient thy-mus anlage fails to grow adequately whencompared to wild-type thymus (22, 42, 144,145). The nude thymus rudiment is also char-acterized by a near absence of hematopoieticcells (22, 42, 146), possibly related to loss ofexpression of the chemokines CCL25 (ligandof CCR9) and CXCL12 (ligand of CXCR4)in the embryonic nude thymus (147). Interest-ingly, embryonic nude thymic epithelial cellsare also decient in expression of the Notchligands Dll1 and Dll4 (145). Although thequestion whether Notch-Notch ligand inter-actions and concomitant T cell commitmenttake place before or after entry into the thy-mus is still debated (see 148 for a recent dis-cussion), lack of Notch ligands in the nudethymus may be prohibitive for T cell devel-opment and, as such, may contribute to thealymphoid nude thymus phenotype.

Virtually all medullary and cortical TECin chimeras constructed from Foxn1+/+ plusFoxn1nu/nu embryos (149, 150) or fromFoxn1+/+ ES cells plus Foxn1nu/nu blastocysts(9) were from the Foxn1+ but not Foxn1nu ori-gin. This demonstrates that Foxn1 acts in acell-autonomous manner in thymic epithelialcells. The action of the Foxn1 gene in cis, withno rescue in trans, implies that the essentialtarget genes of the Foxn1 transcription fac-tor do not include a gene encoding a solublefactor such as a growth factor or a cytokine,or at least none that plays a substantial role inthe nude thymus phenotype. Although geneshave been identied that are differentially ex-pressed between Foxn1+ and Foxn1nu TECs(144), functionally crucial target genes areunknown. This may appear surprising giventhat Foxn1 has been known for some time. It

should be noted, however, that it has been im-possible to study the true function of Foxn1 ei-ther in the development or in the maintenanceof thymus epithelium in cell lines in vitro.This necessitates analyses of primary thymicepithelial cells that are rare cells (on the or-der of a few percent of total thymus cells) ina normal thymus and are even rarer in a nudethymus. Hence, although cell numbers of pri-mary wild-type and mutant TEC are per-missive for genome-wide expression analyses,low cell numbers have precluded biochemicalstudies, including the identication of rele-vant DNA binding sites or protein cofactorsthat regulate the transcription factor activityof Foxn1 in primary TEC. As a consequence,the molecular function of Foxn1 in thymic ep-ithelium remains enigmatic. Likewise, it willbe of interest to determine the role of Foxn1beyond the developmental block in nudemice, that is, in differentiated thymus epithe-lium and perhaps during thymus involution.

Traf6 and RelB

TEC differentiation is completely blocked inFoxn1nu/nu mice at a stage precluding any sup-port of T cell development. In contrast, thy-mus development is permissive in mice de-cient in the NF-B component RelB or theTNF receptor-associated factor Traf6. Never-theless, both of these mutants show major de-fects in TEC structure and function that mayindicate faulty TEC differentiation or mainte-nance. Traf6 is a signal transducer in the NF-B pathway that activates IB kinase (IKK) inresponse to proinammatory cytokines. IKKconverts the RelB/p100 dimer to RelB/p52,which activates target genes in the nucleus(reviewed in 151). Based on an NF-B ac-tivity reporter mouse, the NF-B pathway isused in the developing thymus in cells witha medullary location (152). In the absence ofTraf6 (153), the thymus architecture is disor-ganized and the medulla-cortex separation isblurry. Medullary TEC populations are eithermissing or lack expression of certain cell sur-face markers (UEA-1 but K5+ phenotype).

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These mutants suffer from autoimmunity thatcorrelates strongly with lack of Aire+ TEC,concomitantly reduced expression of tissue-restricted self-antigens on the TEC side, andan absence of Foxp3+ regulatory T cells inthe thymus. These defects are indeed stromalcellintrinsic, as shown by transfer of autoim-mune symptoms by thymus grafting. RelBexpression was markedly reduced in thymusstroma ex vivo and in TEC cell lines and couldbe restored by reintroduction of Traf6. Thediminished expression of RelB is a plausiblemolecular mechanism because earlier workhas shown that RelB-decient mice also havesevere medullary defects that render mice au-toimmune (154156). The phenotype of theTraf6-decient thymus has been proposed tobe independent of LTR ligand signaling onmTEC (118) because LTR-induced activa-tion of NF-B was unaffected by lack of Traf6(153). Overall, the importance of differentNF-B pathways in thymus epithelium is notentirely clear because Traf6-independent NF-B activity was noted in the aforementionedreporter mouse (152).

The molecular defects in mice lacking RelBor Traf6 give novel insights into the geneticrequirements for a key thymus function, tol-erance induction. However, further interpre-tations of these and other studies into TECdefects are complicated by the ambiguity ofwhether the gene defects cause ablation of acell type (e.g., Aire-expressing mTEC) or al-ter the gene expression (e.g., Aire in otherwisenormal TEC).

p63

Expression of p63, a homolog of the tumorsuppressor p53, is required for the normal de-velopment and maintenance of many epithe-lial tissues. The widespread epithelial defectsin mice lacking p63 are consistent with a cru-cial function of this gene in epithelial stemcells. p63/ mice possess very small thymithat were recently analyzed in detail (136,157). p63 was expressed in all K8+ thymicepithelial cells on E12 and continues to be

expressed later in ontogeny in some but notall medullary and cortical TEC. Numbers ofthymocytes were very low, but T cell devel-opment was normal in p63/ thymi. Hence,mutant TEC are, in principle, functional (136,157). This also holds true for hematopoi-etic progenitors, as shown by T cell devel-opment from p63/ fetal liver stem cellstransferred into Rag-2-decient mice. Fur-ther experiments show that TEC from p63/

thymi have reduced expansion potential in ageneral epithelial cell colony assay (136). Us-ing single cell suspensions from rat thymus,Senoo et al. (136) also developed an inter-esting assay for continuous culture of thymusepithelium that was considered a thymus ep-ithelial stem cell assay. Cells that expandedin this assay expressed p63, and knock-downof p63 strongly impaired the colony size, di-rectly supporting the argument for an impor-tant role of p63 in TEC or TEC stem cellmaintenance.

Because support of T cell developmentis the ultimate functional test for TEC, itwould be interesting to know whether TECthat arise in this stem cell assay are func-tional. An additional mechanism for the ab-normally small p63/ thymus may be the re-duced expression of p63 target genes, someof which are familiar as they play a role inthymus organogenesis. Notably, FgfR2-IIIb,which was mentioned earlier as a crucial re-ceptor involved in mesenchymal-epithelial in-teractions (72), as well as Jag2 were downregu-lated (157). Collectively, these recent ndingson the role of p63 in TEC development, andpossibly TEC stem cell maintenance, mayopen new experimental access to the iden-tity of thymus stem cells and their molecularregulation.

FUNCTIONAL CERVICALTHYMUS IN MICE

Incidence

Recently, evidence has been provided thatmice have a cervical thymus that is indeed


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functional in its ability to be colonized bybone marrowderived progenitors, to gener-ate thymocytes that are TCR repertoire se-lected according to the laws of positive andnegative selection, and to export mature im-munocompetent T cells. Terszowski et al. (41)found cervical thymus in chimeras generatedas controls for mutant chimeras initially madeto test the function of Tbx1 in thymus organo-genesis. Dooley and colleagues (158) searchedfor ectopic thymus in the neck based on thesimilarity of thymus epithelium with non-thymic epithelium such as respiratory epithe-lium (90) and on the observation that sam-ples of human parathyroid showed inclusionsof ectopic thymus (158). Both reports pro-vided evidence for a strain-dependent inci-dence of cervical thymi ranging from 50%(158) to 90% (41) in BALB/c mice and thelower but still signicant frequency of 30%

Foxn1:: Egfp

a b

C57BL/6

3 mm 2.6 mm

Neck

Neck

ChestChest

Figure 6Functional cervical thymi in mice. (a) Visualization of bilateral cervical(neck) thymi in a Foxn1::Egfp reporter mouse, and (b) histologicalcomparison of May-Grunwald-Giemsa-stained tissue sections from thethoracic thymus and one cervical thymus lobe in a C57BL/6 mouse. Thefunctional properties of neck thymus have recently been described (41, 158).

(158) to 50% (41) in C57BL/6 mice. Thesedifferences might be explained by the differ-ent counting of either all detectable (41) oronly medially located thymi (158). As othertools of visualization of thymus become avail-able (39, 40, 41), frequencies of cervical thy-mus in mice can be measured more pre-cisely. Examples of cervical thymi are shownin a mouse line (FVB C56BL/6 back-cross) transgenic for a Foxn1Egfp reporter genein which Foxn1-expressing cells are visual-ized specically by green uorescence (41)(Figure 6a), and in a normal C57BL/6 mousein which two thoracic and one cervical lobe aredisplayed as May-Grunwald-Giemsa-stainedtissue sections (Figure 6b). Hence, althoughcervical thymi are not detectable in every sin-gle mouse in the strains analyzed so far, twoof the most commonly used laboratory strains(BALB/c and C57BL/6) frequently have cer-vical thymi. Although the neck region of nudemice has, to my knowledge, not formally beenexamined for cervical thymi, the known lackof functional T cells in this mutant and the ex-pression of Foxn1 in TEC in the neck thymus(41, 158) support the argument for a commondefect in the generation of thoracic and cer-vical thymi in the nude mouse.

Thoracic and Cervical Thymusin Other Mammals

The cervical thymus in mice is not with-out precedent, because neck thymus is knownfrom other mammals. Primitive mammalssuch as marsupials (pouched mammals) pro-vide interesting examples. Most marsupialshave only thoracic thymus, whereas others,interestingly, have both thoracic and cervi-cal thymi (kangaroo or possum), and yet oth-ers have only a cervical thymus (koala) (159).In some of these species, cervical thymus tis-sue can be mingled with parathyroid tissue. Insheep, cattle, and pigs, the thymus has cervicaland thoracic parts belonging to one thymusand therefore they are connected to each other(160). In humans, case reports of disease-associated cervical thymi (161163) suggest

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that cervical thymus is rare (see references in41, 158) and is caused by failure of the thy-mus to properly descend to its nal mediasti-nal location. However, others have estimatedthat the cervical thymus in humans may bequite frequent, with an incidence of 50% inadults (further references in 158) and 60% inchildren (164). Collectively, precise gures onthe normal incidence of neck thymus in hu-man are difcult to extract from the literature(for further references and considerations, seealso 162), and a denitive answer to this ques-tion may require further and more systematicimaging data.

Origin of the Cervical Thymusin Mice and the Relationshipof Thymus and Parathyroid

The following possibilities can be consideredfor the origin of the second thymus.

1. The cervical thymus originates from thethird pouch as the thoracic thymus does,but it takes the parathyroid route of mi-gration. This could occur as a resultof imprecise separation of the parathy-roid and thymus domains (62) withinthe third pouch epithelium, leading tothe partition of thymus progenitors intothe parathyroid anlage and vice versa(see below). This would be reminis-cent of the model of secondary split-ting of the thymus anlagen in chicken(Figure 3). In this regard, it is note-worthy that thymus and parathyroidorgans do not only develop from thesame pharyngeal pouch but also share,at least in mice, some functional sim-ilarities. Some 70% of Gcm2-decientmice survive despite a complete absenceof parathyroids (64). In mice, but not inhumans (165), the thymus was identiedas the auxiliary source of parathyroidhormone (PTH) (64). However, thoughexpression of Gcm2 is restricted to theparathyroid and is required for the de-velopment of precursors for this organ

(104), the PTH-producing cells in thethymus colocalize with Gcm1, a genehomologous to Gcm2, that is expressedin clusters of cells in the thymus (166).The lineage of these Gcm1-expressingcells in the thymus is unknown.

2. The anlage for the cervical thymusarises independently in a differentpouch, and, up to now, these few TECprogenitors may have escaped detec-tion. An origin in a separate pharyn-geal pouch would be akin to the multiplethymus anlagen in the shark (Figure 3)and could be considered an atavism.

3. There could be a delayed epithelialspecication toward the cervical thymusfate at an unknown location in the neck.In this case, a search for cells express-ing Foxn1 between days 14 and 18 out-side the thoracic thymus may providea clue. This latter idea is supported bythe notion that the development of thecervical thymus is delayed by about oneweek compared to the thoracic thymus(41) (see below).

Developmental Kineticsof the Cervical Thymus

The identication of the primitive anlage forthe cervical thymus in the neck o