Organog Timo Ann Review Imm 08

ANRV338-IY26-12 ARI 16 February 2008 12:33
Thymus Organogenesis
Hans-Reimer Rodewald
Institute for Immunology, University of Ulm, D-89070 Ulm, Germany;
email: hans-reimer.rodewald@uni-ulm.de
Annu. Rev. Immunol. 2008. 26:35588
First published online as a Review in Advance on
November 30, 2007
The Annual Review of Immunology is online at
immunol.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 epithelial
stem and progenitor cells, transcription factors, cervical thymus,
reaggregate organ cultures, Cre recombinase, fate mapping, nude
blastocyst complementation
Abstract
The epithelial architecture of the thymus fosters growth, differen-
tiation, and T cell receptor repertoire selection of large numbers of
immature Tcells that continuously feed the mature peripheral Tcell
pool. Failure to build or to maintain a proper thymus structure can
lead to defects ranging from immunodeciency to autoimmunity.
There has been long-standing interest in unraveling the cellular and
molecular basis of thymus organogenesis. Earlier studies gave im-
portant morphological clues on thymus development. More recent
cell biological and genetic approaches yielded new and conclusive
insights regarding the germ layer origin of the epithelium and the
composition of the medulla as a mosaic of clonally derived islets. The
existence of epithelial progenitors common for cortex and medulla
with the capacity for forming functional thymus after birth has been
uncovered. In addition to the thymus in the chest, mice can have a
cervical thymus that is small, but functional, and produces T cells
only after birth. It will be important to elucidate the pathways from
putative thymus stem cells to mature thymus epithelial cells, and
the properties and regulation of these pathways from ontogeny to
thymus involution.
355
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INTRODUCTION
The classical embryological view of thymus
development that had been perpetuated for
decades has undergone major revisions within
the past few years. Advancing the under-
standing of thymus organ development has
been slow by comparison to the progress
in research addressing cellular pathways and
underlying molecular mechanisms of devel-
oping T cells. Why are experiments address-
ing the development of the thymus inter-
esting? The unique function of the thymus
in establishment and maintenance of the T
cell arm of the immune system is intimately
linked 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 is
broadly recognized that thymus structure is
key tothese specic immunological properties
of 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 thymus
organogenesis.
Novel experimental access has been facil-
itated by the development of experimental
tools such as, to name a few, stromal cell isola-
tion by phenotype-based cell sorting (e.g., 7
9), dissociation and reaggregation of stromal
cell 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 in
thymus epithelial cell (TEC) subsets (5). Key
questions in thymus organogenesis surround
the fundamental cellular and molecular mech-
anisms that lead to the formation of a normal
thymus. Areas of interest include the tran-
sient or permanent germlayer contribution to
thymus structure, that is, origin of TEC, and
other stromal elements; the quest for thymus-
building and/or maintaining stem or progen-
itor cells; an understanding of the turnover of
thymus stromal elements in the steady-state
thymus; and the role of thymus stromal cells
in thymus involution or pathology-associated
transient thymus malfunctions (16). In par-
ticular, quantitative answers to many of these
questions would be useful. For instance, the
number of cells with TEC-forming potential
(TEC progenitors) needs to be determined,
as well as their clonogenic potential, steps of
commitment, life span, etc.
Thymus stroma can be viewed as all non-
hematopoietic components of the thymus that
are 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-hematopoietic
marker 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 composed
of two major subsets referred to as cortical
TEC (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 connective
tissue cells, and endothelial cells forming the
typical thymus vasculature (9, 18, 19). Fi-
nally, dendritic cells and macrophages that are
CD45
+
hematopoietic cells are alsoimportant
elements of thymus stroma.
Stroma is not only heterogeneous at a
given time point, but its composition also
varies considerably over time (20). Such re-
structuring can reach extreme forms un-
der thymus-ablating conditions (steroid treat-
ment, irradiation, cachectic conditions) or
with age-associated thymus involution, events
that 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 development
or function of given cell types in the phys-
iological context and their transient or per-
manent contribution to the thymus structure
and function. Moreover, direct from indirect
356 Rodewald
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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 1
Major cell types in the thymus and their developmental origin. The thymus can be divided into
hematopoietic cells (CD45
+
) that are transient passengers and resident stromal cells (CD45
). CD45
cells include two lineages: Thymus epithelial cells (TEC, Keratin

+
) that originate from pharyngeal
pouch endoderm (third pouch in the mouse) and mesenchymal cells (Keratin
), which are a mixture of

cell types that contribute to various structures of the thymus such as capsule or vasculature. The origin of
the mesenchyme appears heterogeneous. The ratio of CD45
+
to CD45
cells is about 50 to 1, but most

of the depicted cell types can be isolated based on phenotype from thymus cell suspensions following
enzymatic 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 drawn
about the relationship between overall organ
architecture and Tcell development, and vice
versa, or, in short, the concept of crosstalk
(21).
Until about 2001, the prevailing view of
thymus organogenesis was, at least in part,
based on early morphological studies that had
to rely on extrapolation from standing pic-
tures to moving cells (22). Anatomical but
also functional data were obtained by graft-
ing experiments in developing birds (23, 24).
Collectively, these studies led to the view that
www.annualreviews.org Thymus Organogenesis 357
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epithelial cells of endodermal and ectodermal
origin (22) and mesenchymal cells of neu-
roectodermal (neural crest) origin (23, 24)
all contributed directly or indirectly to the
thymus. Directly means that cells and their
progeny constitute thymus structure, as is the
case for pharyngeal pouchderived epithe-
lium, and indirectly means that cells provide
inductive signals in trans. Neural crest (NC)-
derived mesenchyme is thought to fall in this
latter category. To what extent it also con-
tributes to thymus structure has not been fully
resolved.
The mechanism that establishes the sepa-
ration into the inner, morphologically lighter
zone, the medulla, and the outer, morpholog-
ically darker zone, the cortex, had apparently
beensettled many years ago. Alook at the evo-
lutionof the immune systemstrongly suggests
that medulla-cortex organization is function-
ally important because, as soon as there was
a thymus, this architectural hallmark of the
thymus 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 was
derivedfroma layer of ectodermal epithelium.
The latter was donated by the cervical vesicle,
ectodermal epitheliumthat comes closely into
the proximity of the endodermal pouch at one
point in ontogeny (embryonic day 10.5 in the
mouse). Hence, medulla-cortex organization
was supposed to be established from two cell
layers by a mechanism involving invagination
and circumferential growth. The model im-
plying cell layer movements was revised by the
nding that the medullary epithelium is com-
posed of single epithelial cellderived islets
that coalesce to formlarger medullary areas in
the adult thymus (13, 26). Hence, the medulla
develops from few progenitors, and the ex-
tent of progenitor proliferationestablishes the
boundaries between medulla and cortex.
The remarkable capacity of cell suspen-
sions of puried fetal thymus epithelial cells
to reaggregate in vitro (11) and to forma func-
tional thymus when grafted under the kid-
ney capsule (12) paved the way to examine
the potential of fetal epithelial cells, or sub-
sets thereof. Based on this approach, a TEC
stem/progenitor phenotype was proposed a
few years ago (14, 15), but the exclusivity of
this phenotype was questioned recently (27).
Hence, the search to identify, enumerate, and
functionally characterize true thymic stem or
progenitor cells is only beginning. In fact, it
is not clear whether self-renewing thymic ep-
ithelial stem cells exist, and if so, to what ex-
tent they are involved in the generation, or
regeneration, of the thymus.
Grafting techniques were further rened
by mixing in single, genetically marked cells
into a donor thymus, followed by grafting
of this tagged tissue, and subsequent visual-
ization of single cellderived medullary and
cortical TEC progeny (26, 28, 29). More-
over, genetic activation of single thymic ep-
ithelial cells in athymic nude mice showed
that one TECprogenitor could formsmall yet
functional thymus units, again composed of
medulla and cortex. This can happen, at least
experimentally, early after birth in the thorax
and hence later than normal and away fromits
normal physiological place, the third pouch
(26; discussed in 29). On the basis of dye-
marking and embryo culture techniques and
in line with earlier studies on thymus devel-
opment in birds (23), researchers abandoned
the dual germ layer origin model of mouse
thymus epithelium in favor of a single endo-
dermal origin model (30, 31). By denition,
the identication of clonal common medulla
andcortex progenitors alsocalls for a common
germ layer origin (26, 28).
The structural components of the thy-
mus have been only poorly accessible for a
long time, mostly owing to difculties or in-
efciency in retrieving these cells from the
solid organ. This, however, is a prerequisite,
for instance, to probe the stromal cell sub-
sets functionally (10) or phenotypically (8).
Monoclonal antibodies and other reagents
that specically recognize subtypes of stro-
mal cells have been instrumental in dissecting
the thymus structure (8, 3236). Mutations
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can now be introduced into thymus epithe-
liumby tissue-specic gene targeting (37) that
has been applied to thymus stromal cells (38,
39), or by nude mouse [ forkhead box N1 gene
(Foxn1
nu/nu
)] blastocyst complementation (9).
Mice expressing Cre recombinase specically
in ancestors and/or their progeny of thymus
epithelial cells (Foxn1
Cre
) (39, 40) will prove
useful not only for studying gene function in
thymus organogenesis but also for clarifying
the origin and lineage relationship of stro-
mal cell subsets (fate mapping). Strategies for
gene targeting in, and fate mapping of, thy-
mus epithelial cells are depicted in Figure 2.
Visualization of thymus epitheliumby expres-
sion of markers such as enhanced green u-
orescent protein (Egfp) (Foxn1
Egfp
) (41) or
enzymes (Foxn1
LacZ
) (39, 40) under the con-
trol of TEC-specic genes should also shed
new light onto the development and mainte-
nance of TEC and reveal temporal and spe-
cial changes in expression patterns of TEC
genes. Finally, genetic screens in vertebrates
other than mice, e.g., zebrash, are ongoing
and aimat the identication of newgenes that
control thymus organogenesis (42, 43).
Rather than attempting a comprehensive
coverage of all current knowledge of thymus
organogenesis, this review focuses on signi-
cant recent advances in the eld, starting with
a brief primer on the embryological origin of
the thymus in phylogeny and concluding with
the recently identied functional second thy-
mus in mice, located in the neck.
PHYLOGENY OF THYMUS
ORGANOGENESIS
The appearance of the thymus in evolution
is linked to the appearance of lymphocytes
expressing highly diverse antigen-recognition
receptors based on DNA recombination of
variable (V), diversity (D), andjoining( J) gene
elements. This diversity, combined with strin-
gent selection processes forced onto devel-
oping lymphocytes, allowed for self-nonself
discrimination 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 anatomical
microcompartments that serve as specialized
hematopoietic and stem cell niches, it is note-
worthy that T cells required an entire organ,
and not merely a niche. The thymus is an
autonomous organ, physically separated from
the general primary hematopoietic sites such
as the bone marrow. The destructive potential
of T cells, once released into the body follow-
ing incomplete or faulty selection in an only
poorly separated niche, could have necessi-
tated the emergence of an entirely separate
organ.
No thymus is known in species more prim-
itive than vertebrates. Among vertebrates,
only jawed, and not jawless (agnatha such as
lamprey), species have a thymus (4446). The
precise embryological origin of the thymus,
the number of thymus organs per animal,
and the nal anatomical positions of thymus
lobes all differ markedly in different species
(Figure 3) (reviewed in 48). The common
theme is that the thymus always originates
frompharyngeal 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, or
head regions, including the thymus, and the
parathyroid gland (49). The origin of the thy-
mus in the inner layer of an embryonic gut an-
cestor is reminiscent of GALT(gut-associated
lymphoid tissue), which is a key lymphoid
structure in species prior to the appearance of
a thymus. Thus, the thymus may have evolved
as a GALTderivative (45). The most primitive
thymus-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 pouch
in frogs, in the second and third in reptiles,
and in the third and/or fourth in bony sh,
birds, and mammals (Figure 3). Thus, species
are exible in positioning of the thymus an-
lage somewhere along the pharyngeal foregut
endoderm. 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 thymus
pairs, while many teleost sh species, anuran
amphibians (e.g., frogs), and many mammals
have only one thymus composed of two bilat-
eral lobes. The position of thymus in the neck
and/or in the chest in different mammals is
discussed in the context of the cervical thy-
mus in the mouse.
In some species, each thymus has a private
anlage. 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
Foxn1
Cre
, Keratin
Cre
, or other loci
Foxn1
nu/nu
blastocyst
Foxn1
+/+
ES cells
with homozygous
mutation in gene
of interest
Advantage
Principle
Disadvantage Nude blastocyst complementation is
laborious because it requires generating
chimeras by repeated blastocyst
injections. In addition, homozygous
null ES cells are required, and the
method is constitutive and not
conditional.
Foxn1
Cre
mouse
Keratin
Cre
mouse
Others
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 Foxn1
Cre
(or Keratin
Cre
) may lead to a mosaic in TEC.
The spatial distribution of deleted versus
nondeleted TEC is a challenge to analyze.
a b
Foxn1
Cre
drives Cre expression in all or
the vast majority of Foxn1-expressing cells
at any time in ontogeny, leading to homozygous
deletion in floxed alleles in mTEC and cTEC.
This also works using Keratin
Cre
mice, or
Cre mice that drive expression of Cre in all
or subsets of TEC.
The Foxn1 gene acts in cis in TEC.
Hence Foxn1
nu/nu
cells cannot be
rescued by Foxn1
+/+
cells in trans.
In Foxn1
+/+
ES into Foxn1
nu/nu
blastocyst chimeric mice, TEC originate
from Foxn1
+/+
ES cells. Hence, TEC
in 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.
360 Rodewald
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primordia each give rise to one thymus lobe
positioned along each side of the body. Thus,
multiple thymi arise in a one-anlage-to-one-
thymus ratio (Figure 3). In contrast, in the
chicken, the anlagen in the third and fourth
pouches give rise to one immature thymus
that subdivides secondarily into multiple in-
dividual thymus lobes positioned along the
neck (Figure 3) (50). As we begin to think in
terms of organ progenitor cells, it is likely that
the original pharyngeal anlage in the chicken
harbors a certain number of progenitor TEC
that are partitioned into separate cell clusters,
each of which gives rise to one nal thymus
lobe.
CELLULAR BASIS OF THYMUS
ORGANOGENESIS:
ENDODERMAL EPITHELIUM
AND THE GERM LAYER ORIGIN
OF THE THYMUS
Inanalogy togeneral organdevelopment, thy-
mus organogenesis has been divided into sev-
eral consecutive steps: (a) positioning; (b) bud-
dingandoutgrowthof the thymus anlage from
the third pouch; (c) detachment of the prim-
itive thymus from its endodermal basis; and
(d ) patterning, differentiation, and migration
of the thymus toward its nal anatomical posi-
tion (51, 52). Positioning refers to pouch for-
mation at the prospective site where epithelial
cells will later undergo commitment toward
thymus and parathyroid fates. Morphological
three-dimensional reconstructions (22) indi-
cate that, on embryonic day 9 (developmental
timing is fromanalyses of the mouse), the pha-
ryngeal pouch constitutes a double-layered
membrane composed of an ectodermal and an
endodermal cell sheet. Onday 9.5, these layers
blend together, and it is likely that the precise
germ layer origin of these epithelial cells in
the thirdpouchcannolonger be assignedwith
certainty solely based on morphology. Later,
on day 10.5, parts of the ectodermal cervical
vesicle come into close contact with the en-
doderm of the third pouch. It was thought
that these ectodermal cells rapidly proliferate
and nally surround the endodermal tissue.
These and earlier (53) observations formed
the basis for the long-held textbook view (54
56) of a double germ layer origin of the thy-
mus with endodermal and ectodermal origins
of medullary and cortical epithelium, respec-
tively. In contrast, on the basis of grafting ex-
periments in birds, researchers concluded that
thymus epithelium is derived from a single
germ layer, the endoderm (23, 24), that re-
quired the presence of NC mesenchyme (see
below).
More recently, the question of the germ
layer origin of thymus epithelium was read-
dressed, this time in the mouse (30). Examina-
tion of the histiogenesis of the thymus during
the critical period (E10.5 to E12) conrmed
that third pouch endoderm and third cleft
Figure 2
Strategies for gene targeting and fate mapping of thymus epithelium. (a) Mutations can be targeted to the
thymus epithelium, and gene function can be analyzed in TEC by the generation of chimeric mice made
by injection of Foxn1
+/+
embryonic stem (ES) cells into nude (Foxn1
nu/nu
) blastocysts, termed nude
blastocyst complementation (9). This strategy takes advantage of the fact that all thymic epithelial cells in
such chimeras (9), as well as in aggregation chimeras of Foxn1
+/+
and Foxn1
nu/nu
embryos (149, 150), are
derived 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 thymus
in which TEC lack the gene that is deleted in the ES cell. Nude complementation has been used to study
the role of vascular endothelial growth factor (Vegf )-A in thymus epithelium, a gene that is heterozygous
lethal if mutated in all cells of an embryo. (b) Thymus epithelium can be genetically modied using
TEC-specic Cre recombinase deleter mice such as Foxn1
Cre
(40), or various Keratin
Cre
(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 the
experiment will be. Advantages and disadvantages are listed.
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i m y h T s e h c u o p l a e g n y r a h P
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 3
Origin of the thymus in phylogeny: a variation of the theme. A
phylogenetic comparison of the origins of the thymus in shark (a),
chicken (b), and mouse (c) demonstrates that thymus anlagen can be found
in different pharyngeal pouches (pp) and that the ratio of mature thymus
lobes per pharyngeal pouches can differ. In the shark, each thymus lobe
has its own anlage (a), whereas the chicken splits one thymus, from two
pouches, into seven lobes (b). In the mouse, the thoracic thymus
originates from the third pouch. It remains to be determined whether the
cervical thymus in mice develops akin to the chicken or the shark thymus.
ectodermindeedmake contact. Evidently, this
contact does not result in a compound struc-
ture incorporating cells of both germ layer
origins. Instead, there are signs of apoptosis
at the contact site, suggesting a cell loss, pre-
sumably on the ectodermal side (30). More-
over, when the outer pharyngeal surface of
E10.5 embryos was dye-labeled in vitro and
these ectodermally tagged embryos were cul-
tured for 30 h, there were no labeled cells
found in the thymus. This indicates, again,
that ectodermal epithelium does not con-
tribute to the thymus proper. In addition to
these experiments involving in vitro dye la-
beling and embryo culture techniques (57),
endoderm-only third pouch tissue was dis-
sected fromE9 embryos and grafted into nude
recipients. These grafts developed into func-
tional thymi with medulla-cortex architecture
(30).
There is also genetic evidence for a
common germ layer origin of cortical
and medullary epithelium from tetraparental
chimeric mice generated by injection of ES
cells into MHC-mismatched blastocysts (13).
There was no clear relationship between the
origin of mTEC islets, discussed later in the
context of TEC progenitors, and the ori-
gin of the cortex immediately surrounding
a medullary islet (13). In other words, a
medullary 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 mTECand
cTEC, might imply that, overall, the origins
of mTEC and cTEC are proportional. The
relative contribution of the ES cell and the
blastocyst to the tested organs (thymus, skin,
liver, heart) was random, suggesting that these
organs developed independently (Figure 4).
In contrast, comparison of the overall contri-
bution of ES cell or blastocyst to medullary
and cortical epithelium revealed a straight
line pointing at a common origin and close
362 Rodewald
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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 for
the entire thymus epithelium. Collectively,
independent experimental approaches have
now 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 fromthe third pouchcoincides withonset
of expressionof the Foxn1 gene (60). Foxn1 ex-
pression is rst detected in a subset of epithe-
lial cells in the third pouch on embryonic day
11.5 in mouse development (6163). Double-
staining for expression of Foxn1 versus glial
cells missing 2 [Gcm2], a transcription factor
gene required for parathyroid organogenesis
(64), reveals adjacently located epithelial cell
clusters whereby Foxn1
+
cells are located in
the ventral part of the third pouch while Gcm2
marks the dorsal aspects of the same pouch
(62). Because Foxn1 and Gcm2 are essential for
thymus (60, 61) and parathyroid (64) devel-
opment, respectively, these cell clusters likely
represent the anlagenfor eachor the two third
pouchderived organs. However, a direct and
exclusive precursor-product relationship be-
tween Foxn1- or Gcm2-expressing cells in the
pouch and the mature organs has not been
established. This would require the purica-
tion of cells based on their expression of these
transcription factors and a prospective test of
their potential.
CONTRIBUTION OF NEURAL
CREST (NC) MESENCHYME TO
THYMUS ORGANOGENESIS:
TRANSIENT OR LONG
LASTING?
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 this
phenomenon 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
S
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x
Medulla
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Skin
100
100
50
50
0
0
Figure 4
Common origin of medullary and cortical TEC in tetraparental (chimeric)
mice. Mice generated as described by injecting embryonic stem (ES) cells
into MHC-mismatched blastocysts (13) were analyzed for ES cell versus
blastocyst contributions to skin, heart, liver, and thymus utilizing
microsatellite differences in ES cell and blastocyst genomic DNA. The
contributions of ES cell and blastocyst to thymus medulla and cortex were
determined by histological analysis of the MHC class II haplotypes
indicative of ES cell or blastocyst origin. None of the organs showed any
linkage of the origins except for thymus cortex and medulla.
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thymic epithelium, and forms perivascular
mesenchyme (23). Ablation of cephalic NC
in birds prevented any contribution to or in-
duction of thymus epithelium by NC-derived
mesenchyme, and this resulted in small thymi
with delayed development and poor function
(24). These early reports were conrmed in
principle and extended by many subsequent
experiments that showed an important role
for NC in thymus development in vivo (re-
viewed in 51, 52) and in organ culture sys-
tems (10, 67, 68; reviewed in 1). Examples
of genetic pathways involved in mesenchyme-
thymus epithelium interactions are provided
below.
Given that mesenchymal-epithelial con-
tact takes place early in development and that
mesenchymal cells are abundant in the adult
thymus, as specied in the introduction, it has
been debated whether or not embryonic NC-
derived mesenchyme and adult thymus mes-
enchyme share a precursor-product relation-
ship (reviewed in 66). Several reports address
this point in mice generated with the aim to
express Cre recombinase specically in the
NC lineage. In combination with appropriate
Cre activitydependent reporter mice, NC-
derived cells and their progeny should be per-
manently labeled in this system. Mesenchyme
marked by Wnt1
Cre
surrounds the E13.5 thy-
mus as a massively stained, thick layer. How-
ever, labeled cells become rare as soon as
the thymus grows, owing to the rapid prolif-
eration of thymocytes. In the adult thymus,
the overall contribution of Wnt1
Cre
-marked
cells appears too low to account for major
mesenchymal 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). A
similar result of transient but not permanent
participation was obtained using a different
NC-specic marker gene, myelin protein zero
(P0). By ow cytometry on thymus cells from
E13.5, as many as 30% of all stromal cells are
labeled by P0
Cre
(70), which is the rst quan-
titative estimate of the contribution of NC to
the developing thymus. Again, few marked
cells persist at later stages. In vitro, P0
Cre
-
marked cells from the fetal thymus could be
developed into melanocyte and glial cell lin-
eages, further supporting their NC origin.
The idea that the thymus contains cells related
to neuronal/glial lineages is not new (71), but
there is currently no evidence to dene func-
tions for such cells in the thymus. Finally, a
cautionary note may be warranted here be-
cause, as in other fate mapping experiments,
the result will ultimately depend on the strat-
egy used. Randomtransgenic integration may
yield more mouse-to-mouse variability than
targeted knockin approaches, and the result,
here low or no contribution of NC cells to
the adult thymus, depends on the properties
of any particular Cre driver mouse and the
sensitivity 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 growth
factor [KGF]) and their receptors (FgfR).
Fgf7 and Fgf10 are expressed by the mes-
enchyme surrounding the embryonic thymus
epithelium, and the latter expresses FgfR2-
IIIb. Defects in this signaling pathway per-
turb thymus development (72), demonstrat-
ing a growth-promoting role for mesenchyme
toward thymic epithelium. Signals via Fgfs
also induce TEC proliferation (73, 74) and
protect thymus epithelium from injury by
irradiation (75) or by conditions of graft-
versus-host disease (76). Another role of thy-
mus mesenchyme could be the presentation
of growth factors such as IL-7 or c-kit ligand
to thymocytes, but currently no data support
this idea.
GENES AFFECTING THYMUS
DEVELOPMENT PRIOR TO
THYMUS SPECIFICATION
Mutations in a number of genes, including
Hoxa3 (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
364 Rodewald
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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 function
causes pleiotropic defects in embryonic devel-
opment. It is, therefore, often difcult to dis-
tinguish the primary function of these genes
in thymus organogenesis, e.g., in TEC pro-
genitors, from an upstream function, e.g., in
formation or patterning of pharyngeal struc-
tures or in NC migration. It is obvious that
the thymus cannot develop normally if the
third pouch is absent as a scaffold in which
TEC progenitors arise. The effects of such
mutations are therefore upstream of thymus
organogenesis itself. Moreover, it is also pos-
sible that some of these genes are required
both upstream of organogenesis and later in
the thymus epithelium itself. Along this line,
Hoxa3 (90), Pax1 (82), and Pax9 (91) are ex-
pressed in thymus epithelial cells. Because this
was mostly measured by RT-PCR in cell pop-
ulations, frequencies and phenotypes of TEC
expressing these genes are unknown. TEC-
specic deletion of such ubiquitous pathway
genes will be requiredtoresolve their function
in thymus development beyond the general
defects that may be nonspecic for the thy-
mus. An example of TECtargeting was block-
ing of Bmp signaling in TECby expression of
Noggin under the control of the Foxn1 pro-
moter. Whereas blocking of Bmp signaling in
premigratory NC by transgenic expression of
Noggin in NC affected thymus development
indirectly (92), Foxn1-driven inhibition of the
Bmp pathway demonstrated, in addition to
the NC defect, a role for Bmp signaling in-
trinsic in TEC (93). This is compatible with
expression of Bmp family members in the thy-
mus epithelial anlage (94).
Another interesting gene that belongs to
this gene category, and that is related to
DiGeorge syndrome (9598), is the T box
gene Tbx1. DiGeorge syndrome is caused
genetically by heterozygous deletions within
chromosome 22q11 and clinically by malfor-
mations of pharyngeal arch arteries (cardiac
outowtract) and heart, parathyroid hypopla-
sia, and absence or ectopic location of the thy-
mus (96, 99). Hallmarks of this phenotype are
recapitulated in mice lacking Tbx1 (88, 100),
a gene that is located in the deleted region
in humans (88, 100). Tbx1
/
mice display
agenesis of pharyngeal pouches 24 and con-
comitant loss, or malformation, of pharyn-
geal pouchderived organs and tissues (thy-
mus, parathyroid gland, cardiac outowtract)
(88, 100). Tbx1 is expressed in the pharyngeal
pouch endoderm but also in the core meso-
derm of the pharyngeal apparatus and the
pharyngeal ectoderm but not in NC-derived
mesenchyme (101). Thus, Tbx1 expression
may play distinct roles in different anatom-
ical sites (e.g., endoderm versus mesoderm)
during development. Pharyngeal pouches fail
to develop in mice in which the Tbx1 de-
ciency is restricted to the endoderm by means
of preferential deletion in pharyngeal endo-
derm using another Fox family gene locus ex-
pressing Cre, Foxg1
Cre
. Hence, this mutant
recapitulates the defects known fromthe con-
stitutive null mice, including absence of the
thymus (102). This demonstrates that Tbx1
expression in the pharyngeal endoderm is re-
quired for thymus development. However,
mice that lack Tbx1 expression selectively in
the pharyngeal mesoderm, but not the endo-
derm, also have a hypoplastic pharynx with
impaired pharyngeal endoderm and lack a
thymus (103). Conditional reversion from a
defective to a functional Tbx1 allele in pha-
ryngeal mesoderm, but not endoderm, is suf-
cient to rescue major defects knownfromthe
Tbx1 null phenotype (pharyngeal patterning,
cardiovascular defects) but does not restore
thymus development (103). In conclusion, ex-
pression of Tbx1 both in the pharyngeal core
mesoderm and in the pharyngeal endoderm
is a prerequisite for thymus development.
Within the third pouch, Tbx1 expression co-
incides rather with the parathyroid than the
thymus anlage (104), and it remains to be de-
termined if Tbx1 is expressed in TEC and
whether it plays a role in their development.
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EPITHELIAL PATTERNING
AND DIFFERENTIATION,
AND CROSSTALK BETWEEN
THYMOCYTES
AND EPITHELIUM
Coinciding with hematopoietic colonization
that initially occurs around E12 and prior
to 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 compartmentalization
is 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 keratin
family. Major populations of adult mTECand
cTEC have been distinguished by their ker-
atin (K) 5
+
K8
and K5
K8
+
phenotypes, re-
spectively (107, 108 and references therein).
This dichotomy is, however, not absolute be-
cause K5 is also expressed in some cTEC, and
K8 is also found in some mTEC (107). Re-
garding the onset and pattern of K5 and K8
expression in ontogeny, third pouch epithe-
lium at E11.5 was reported as K5
K8
+
(15,
108), whereas others found coexpression of
K5 and K8 already at this stage, even with
high expression of K5 (14). Aprominent TEC
population coexpresses K5 and K8 on days 12
and 13 (14, 15, 108). Using these and other
markers, so-called double-positive TEC are
thought of as progenitors of mature single-
positive K5
+
K8
medullary and K5
K8
+
cor-
tical TEC phenotypes (108110). This idea
was based on the fact that K5
+
K8
+
cells pre-
cede the appearance of mature K5
+
K8
and
K5
K8
+
TEC in ontogeny. Moreover, large
clusters of K5
+
K8
+
TEC are maintained in
mutants with massive early blockade in T cell
development such as compound Kit
W/W
and
common chain ( c
) (111), or RAG2
/
c
mice (thymus stromal phenotypes reviewed

in 105). Thirdly, and further discussed in the
context 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 of
Foxn1 in the epithelium but is independent
of hematopoietic colonization.
The transition from immature TEC phe-
notypes, such as abundant K5
+
K8
+
cells, to
the full medulla-cortex organization is per-
turbed in mice in which Tcell development is
blocked at immature stages. That the TEC
architecture is somehow inuenced by the
presence or absence of particular stages of
thymocytes development has been viewed as
an interdependence between thymocytes and
stroma (36, 107, 112) and has been referred
to as crosstalk (21). In the original case, it was
suggested that the thymus stroma was perma-
nently damaged unless it had proper contact
with developing pro-T cells (CD44
+
CD25
)
and that this contact needed to take place
at fetal stages of TEC development (112).
The transgenic mouse (hCD326tg) onwhich
these conclusions were based expressed 40
60 copies of a human CD3 transgene (113,
114). T cell development in this mouse was
blocked at an early CD4
CD8
(double-
negative) stage prior to expression of CD25,
and numbers of residual thymocytes were very
low. This paucity of thymocytes and the se-
vere block in thymocyte development were
suspected as the cause of aberrant adult TEC
structure characterized by poorly discernable
cortex and an abundance of cells coexpress-
ing K5 and K8 (107, 112). Subsequent studies
found, however, that the hCD326tg thymus
was not simply devoid of thymocytes, but har-
bored B cells (114). Hence, it cannot be ex-
cluded that it is not the absence of developing
thymocytes that perturbs thymus organogen-
esis but the aberrant B cells or their develop-
ment that contributes to the adult stromal cell
phenotype. This interpretation would ques-
tion the specicity of crosstalk between pro-T
cells and TEC.
Another concern regarding models of
crosstalk in thymus organogenesis has been
the inability to distinguish alterations in the
epithelial compartment owing to lack of
366 Rodewald
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signals from developing thymocytes to TEC
from epithelial reactions to a state of inactiv-
ity. The latter scenario could lead indirectly
to defects in the maintenance of the epithe-
lial cells. Along this line, a recent reevalua-
tion of the developing thymus in hCD326tg
mice found normal proportions of immature
K5
+
K8
+
, as well as single-positive K5
+
K8
and K5
K8
+
TEC when compared to wild-
type thymus (115). These data imply that thy-
mus organogenesis may be quite normal in
hCD326tg mice andthat the architectural al-
terations seen in this mutant are secondary to
regular TEC development, a conclusion that
would support the argument against a role of
pro-T cellmediated signaling for the devel-
opment of thymic epithelial cells (115). The
suggestion that the thymus stromal cell archi-
tecture is damaged at long-term unless pro-
T cells contact the epithelium at the proper
time in ontogeny is also contradicted by ex-
periments made in Kit
W/W
c
mice, a mutant
in which thymocyte development was com-
pletely abrogated (116). The severely dysmor-
phic thymus structure of this mutant could
be reverted to a structurally normal and func-
tional thymus when grafted postnatally into a
recipient that provided wild-type hematopoi-
etic stem cells (111). Finally, the fact that the
human thymus of severe combined immuno-
deciency (SCID) patients can be reconsti-
tuted by transplantation of normal bone mar-
row stem cells (117) further supports the
argument against continuous stroma defects
caused by a block in T cell development.
Collectively, much speculation has sur-
rounded the interesting crosstalk concept. A
crosstalk mechanism should involve cell sur-
face molecules such as receptor-ligand pairs
that can transmit signals in both directions,
and the absence of such signals might cause
a specic phenotype on the nonreceiving
end. Some molecularly dened cases for bidi-
rectional crosstalk between thymocytes and
thymus epithelium exist. One documented
example of a crosstalk mechanism from
thymocytes into epithelial cells is the defect
in the mTEC compartment in mice lacking
components of the TNF-TNF receptor
family. Expression of the lymphotoxin-
receptor (LTR) on thymocytes and of a
LTR ligand on mTEC are required for
normal cellularity and architecture of mTEC.
Mutations that interrupt this signaling path-
way lead to structural defects associated with
faulty selection and autoimmunity (118).
Moreover, the recent nding that expression
of RANK ligand on a CD4
+
CD3
inducer
cell population promotes the maturation
of RANK-expressing CD80
Aire
mTEC
progenitors into CD80
+
Aire
+
mTECs (119)
can also be viewed as a form of crosstalk,
albeit between a highly specialized and rare
lymphoid cell and a specic stage of TEC.
There are other candidate molecules that
could 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-bound
ligand [Kit ligand (KL), or stem cell factor
(Scf )] is expressed on stromal cells (120). Kit
signaling into thymocytes via expression of
KL in TEC is crucial for T cell develop-
ment (121), and KL can indeed signal into
epithelial cells (122). However, T cell devel-
opment was permissive in a KLmutant (Sl
17H
)
(H.-R. Rodewald, unpublished data) in which
the cytoplasmic tail of KL is nonfunctional
(123). Hence, there is currently no evidence
for KL-mediatedcrosstalk inthe thymus. Fur-
ther potential examples are Notch and Notch
ligand pairs. Notch-1 is expressed on pro-
and pre-T cells, and Notch ligands, includ-
ing Delta-like (Dll)1 and Dll4, are expressed
in thymus epithelium (reviewed in 124, 125).
Dll1 or Dll4 could signal into the epithelium
and induce changes in the microenvironment
that could ultimately be involved in the thy-
mus phenotype of Notch-1 mutants, includ-
ing an abundance of B cells instead of T cells
in the thymus (124, 125). However, it is not
known whether Notch-Notch liganddriven
mechanisms play a role in crosstalk or in TEC
development, as has been speculated (28).
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POTENTIAL, FREQUENCIES,
AND PHENOTYPES OF
THYMUS EPITHELIAL
STEM/PROGENITOR CELLS
Progenitors for mTEC
Measurements of stem or progenitor cell ac-
tivity, and the prospective isolation by phe-
notype and subsequent analysis of progenitor
potential, ultimately require clonal assays. Al-
though single cellbased assays to study TEC
development in vitro are still lacking, cellular
and genetic strategies have been developed to
follow the fate of single TEC progenitors or
their activity in vivo (13, 26, 28). Clonal pro-
genitor activity for TEC was initially iden-
tied for the medullary lineage in chimeric
mice 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 was
based on techniques that allow disassembly of
embryonic or fetal thymus by enzymatic di-
gestion, purication of stromal cells or subsets
thereof, and reassembly into thymus reaggre-
gates in vitro (10). These RFTOC are func-
tional in that they support Tcell development
in vitro, but the TECarchitecture of RFTOC
in vitro is quite different fromthat of a normal
thymus in vivo.
In RFTOC assembled from puried TEC
in the absence of thymocytes, distinct mTEC
or cTEC phenotypes were present, but these
cells did not form medulla-cortex structures.
In contrast, RFTOC grafted into recipient
mice not only supported steady-state T cell
development, but also achieved proper thy-
mus architecture, including regular medulla-
cortex organization (12, 126). This remark-
able capacity of TEC to build functional
thymus architecture from reaggregated cell
suspensions suggestedthat mTECandcTEC,
randomly arranged in the reaggregates in
vitro, could migrate within the graft to seg-
regate into cortex and medulla. Such sorting
out would have required active migration, or
at least directed movement, and recognition
of the neighboring cells as mTEC or cTEC.
However, experiments using RFTOC assem-
bled fromtwo distinct donor strains, followed
by 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 mTEC
progenitor activity were visible in the form
of epithelial medullary islets, the majority of
which were either of one or the other, but
not of mixed, origin (13). Medullary epithe-
lial islet formation also occurred during nor-
mal thymus organogenesis in vivo, as shown
in chimeric mice made by injection of ES cells
into MHC-mismatched blastocysts. Here, in-
dividual epithelial islets stemmed from either
ES cell or blastocyst origin. Hence, medullary
islets arise from single progenitors. With age,
the islet-like character of mTEC is harder to
recognize, as many mTEC islets coalesce to
form conuent regions of medullary epithe-
lium. The existence of clonal medullary islets
was recently conrmed genetically (26) (see
below), and the principle of islet formation
in chimeric thymus grafts has been used as
an assay to assess the potential of phenotypi-
cally dened embryonic mTEC subsets (127)
or the role of MHC class II expression for
the selection of Foxp3
+
regulatory T cells on
medullary epithelium (128).
Serial sectioning showed that one thymus
lobe of a mouse at two weeks postnatal con-
tained 300 medullary areas (according to
this denition, a medullary area is larger than
a medullary islet). These medullary areas were
composed of one to three islets. Hence, up
to 900 mTEC progenitors are sufcient
to generate the entire medulla in one thy-
mus lobe of a mouse (13). This estimate is
in a strikingly similar order of magnitude as
the estimated 1500 TEC in the E13.5 thy-
mus that express the tight junction proteins
claudin (Cld)-3 and 4 (Cld3,4) (127). Already
on E10.5 in development, Cld3,4
+
cells were
found in the apical epithelial layer in the third
pouch thymus anlage. Subsequently, Cld3,4
+
368 Rodewald
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cells showeda clusteredarrangement that par-
tially overlapped with general mTEC mark-
ers such as MTS10 or UEA-1. In the adult
thymus, Cld3,4 expression marked a subset
of mTEC expressing the autoimmune regu-
lator gene Aire (127). Cld3,4
+
Aire
+
cells are
presumably fully mature mTEC that are busy
in promiscuous expression of tissue-restricted
self-antigens (TRA) (reviewed in 5). By ow
cytometry, several TEC subpopulations were
identied and separated based on Cld3,4
+
and UEA-1 expression. Interestingly, Cld3,4
+
cells, irrespective of UEA-1 expression, gave
rise toonly mTECandnot cTECwhenmixed
into RFTOC and grafted into nude mice,
whereas Cld3,4
low
UEA-1
cells gave rise to

both mTEC and cTEC. These data suggest
that commitment to the mTEC lineage can
take place already by E13.5, at least in cells
dened by Cld3,4 expression. Collectively,
the very early and restricted expression of
the tight junction proteins of Cld3,4 marks
an mTEC pathway. Because the medullary
clusters that arise from Cld3,4
+
progenitors
are very similar in size and frequency to the
medullary islets described earlier, it is possi-
ble that Cld3,4 expression marks the entire
mTEC pathway at some point in the differ-
entiation tree of TEC.
It is not clear whether mTEC progenitors
are only active during ontogeny, or whether
they continue to generate mTEC de novo in
the 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 later
maintained. However, several reports recently
found TEC proliferation indicating rapid ep-
ithelial turnover in the steady-state thymus (5,
20, 129). There are considerable differences in
the reported proliferation rates, ranging from
as 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 mTEC
incorporating BrdU after one week of label-
ing (129). This would translate into half-lives
of about six days in one case versus four weeks
in the other case; the latter is similar to a third
estimate of six weeks (5).
It is obvious fromthe above considerations
that there are large gaps in our understanding
of the sequence of events during mTEC
differentiation from mTEC progenitors to
mature mTEC. Specically, the relationship
of mTEC differentiation stages to mTEC
function (TRA expression and presentation;
full maturation as antigen-presenting cells)
and mTECturnover (proliferation versus cell
death) are only poorly understood. On the
one hand, islets of mTEC vary in diameter
from a minimum of 60 40 to a maximum
of 170 170 m and harbor between 5 and
45 epithelial cells in a two-dimensional lattice
(13). On the other hand, numbers of cells
expressing a particular TRA in the medulla
appear even lower than the cells per islet (129,
130; reviewed in 5). Because an mTEC islet
is originally made by a single progenitor, cells
within an islet might be further diversied
into subclones (131). It is not known whether
this diversication is stable or whether
individual mTEC change their expressed
TRA pattern over time. It will be necessary to
better dene stages of mTEC differentiation
from mTEC progenitors via immature to
mature mTECand to shed light on the signals
that regulate this developmental progression
as well as on the capacity of TEC at each
stage to present antigens and ultimately guide
TCR repertoire selection on thymocyte
populations (5, 119, 129, 131, 132).
Common mTEC and cTEC
Progenitors
It was speculated early on that mTEC and
cTECshare a common progenitor (109, 110).
The idea was based on coexpression of mark-
ers such as keratins on TEC early in thymus
organogenesis, as discussed above in the con-
text of the germ layer origin. More recently,
the generation of a complete and functional
thymus 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 embryonic
day 12.5 (14) or fetal day 15.5 (15) TEC led
to the formation of a thymus composed of
both 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) argued
strongly in the same direction. These poly-
clonal (bulk) experiments in fact demonstrate
that the populations of cells used to assem-
ble the grafts contained all the cells required
for a functionally and structurally normal thy-
mus. What they couldnot answer was whether
or not a common progenitor for mTEC and
cTEC existed and, if so, whether it was re-
sponsible for the generation of both mTEC
and cTEC in the grafts.
This key property of a common progen-
itor, clonogenicity, has only recently been
addressed (26, 28; reviewed in 29). Single ep-
ithelial cells isolated from yellow uorescent
protein (YFP) expressing embryonic E12.5
thymus were injected into a nonuorescent
host thymus, and such single celltagged
thymi were transplanted into recipient
mice to allow for full thymus development.
Immunohistological analysis showed that, in
each case of positive reconstitution, the single
embryonic epithelial cell had produced both
mTEC and cTEC. Because the donor cells
were puried via expression of a pan-TEC
marker [EpCAM1; antibody G8.8 (34)], no
particular TEC subset was selected, implying
that a large proportion of epithelial cells in
the day 12 thymus has dual potential for
mTEC and cTEC and that their progeny
persists in the adult thymus (28).
A different approach addressing progen-
itor activity in thymus organogenesis was
based on epithelial cell tracing using genetic
in situ labeling (26). Cre recombinase under
the control of the humanKeratin14 promoter
(K14
Cre
) effectively acted as a random and
very rare switch that turned on YFP expres-
sion in TEC. Although no labeled cells were
found in the thymus at birth, numbers of mice
with labeled cells and numbers of YFP
+
TEC
per thymus increased with age after birth. At
all times analyzed, labeled progeny remained
very rare, consistent with Cre-mediated YFP
expression only in single or in very few TEC
progenitors. Three patterns of progeny were
noted: (a) mTEC clusters only, reminiscent
of medullary islets; (b) cTEC clusters only;
or (c) mTEC plus cTEC progeny (26). These
results strongly suggest that K14
Cre
randomly
marked TEC progenitors and their progeny
and that this labeling event could occur over
developmental stages that coveredcommonto
committed TEC progenitors. If the genetic
hit occurred at an early postnatal age, TEC
progenitors, endowed with the listed poten-
tialities, persist at least until that age in a nor-
mal thymus. After the putative mTEC versus
cTEC branch in TEC differentiation, cells
probably migrate considerable distances. This
is evident from the space that was observed
between mTEC and cTEC progeny of com-
mon origin in the adult medullary and cortical
zones, respectively (26). The notion of TEC
migration, or perhaps passive movement, dur-
ing development would also t the topolog-
ical dissociation of mTEC versus cTEC of
the same origin in chimeric mice (13). Col-
lectively, based on cellular (13, 28) and ge-
netic (26) evidence, the thymus harbors TEC
progenitors 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 information
on the total number of common mTEC and
cTEC progenitors that normally engage in
the building of a thymus.
The marking of common TEC progeni-
tors left unanswered whether or not single
TECprogenitors are capable of formingfunc-
tional thymus units. This was addressed again
in the K14
Cre
mouse, but in this case the Cre
recombinase was used to revert a nonfunc-
tional Foxn1 allele to a functional Foxn1 al-
lele akin to correcting the nude mutation in
the thymus in vivo. The Foxn1
nu/nu
thymus is
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composed of epithelial cysts that do not sup-
port T cell development. When crossed to a
Cre-dependent LacZreporter, K14
Cre
marked
rare TECthat, in this case, were located in the
wall of the nude thymus cysts. When Cre re-
verted the loss-of-function Foxn1 allele back
to a functional Foxn1 allele, an event that
occurred again randomly in single postnatal
epithelial cell precursors, small units of thy-
mus tissue developed (26). These neo-thymi
showed all tested hallmarks of a normal thy-
mus, including medullary and cortical orga-
nization and expression of Aire. Mice bearing
such thymi had elevated numbers of immuno-
competent T cells that, unlike the few T cells
found in nude mice, expressed a diverse TCR
repertoire. These experiments demonstrate
that the block that occurs in TEC develop-
ment in the nude mouse does not lead to a
complete loss of TEC progenitors. Rather,
TEC progenitors may enter a stage of dor-
mancy or may be continuously generated de
novo in nude mice. Once genetically reverted
to wild type, they can recapitulate normal on-
togeny and complete their differentiation into
functional TEC. It is noteworthy that this de-
velopment can take place outside of the physi-
ological location, the third pouch, because the
cystic thymus rudiments in the nude mouse
are at this age in the chest. For anatomical
and kinetic reasons, it is unlikely that the in-
ductive signals that are providedtothe thymus
during normal ontogeny, e.g., by NC-derived
cells, are available to those thymi that use their
second chance (29). This lack of proper con-
text could at least in part be responsible for
their small size (26).
TEC Progenitor Phenotypes
In the aforementioned single cell exper-
iments, TEC progenitor cells have not
been physically puried except by the pan-
epithelial marker EpCAM1 (28). Are there
cell surface markers known that identify TEC
progenitors? 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 marker
for embryonic TEC progenitors, a proposal
that met with some interest (133, 134). As
noted before, RFTOC assembled from em-
bryonic (14) or fetal (13, 15) TEC developed
into functional thymi in vivo. Although ear-
lier studies showed thymus development in
RFTOC grafts made from several hundred
thousand fetal TEC dened as MHC class
II
+
CD45
cells (12, 13), others reported thy-

mus formation using much lower numbers of
cells using the MTS24 marker for positive pu-
rication. Twelve thousandve hundredTEC
from E12.5 (14), or 2,500 from day 15.5 fe-
tal (15), contained all the cells required for a
thymus. Even when only 500 cells were trans-
planted, nude recipients showed signs of tran-
siently functional thymus (14).
In analogy to other progenitor systems, it
can 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 the
endodermal 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 express
MTS24. By owcytometry, essentially all thy-
mus epithelial cells, gated as pan-cytokeratin
+
or EpCAM1
+
cells on E12.5 and 13.5, are
MTS24
+
(27), suggesting that at this stage of
development, MTS24
cells are nonepithe-

lial cells. Consistent with these data, and not
surprisingly, only MTS24
+
(that is, epithe-
lial) and not MTS24
(that is, nonepithelial)

cells from E12.5 have thymus-forming capac-
ity (14). Is MTS24a progenitor marker at later
stages of thymus development? On day 15.5,
about half of all TEC still express MTS24
before the frequency of MTS24
+
cells de-
clines to a few percent at later fetal and adult
stages (14, 15, 27, 135). One report found
thymus potential exclusively in MTS24
+
and
not MTS24
epithelial from day 15.5 thymus

(15). However, a recent reassessment of TEC
progenitor activity using MTS24 expression-
based purication did not reproduce these
ndings using larger cell numbers from
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14- or 16-day-old thymi and concluded that
both MTS24
+
and MTS24
epithelial cells
were similarly potent in forming a functional
thymus (27). The available evidence suggests
the following order of events: MTS24 is ini-
tially a nonthymus-specic marker of pharyn-
geal endoderm cells; it then marks all TEC
around day 12.5 and is expressed on day 15.5
on a major subset of TEC. At this stage, thy-
mus potential is included in both MTS24
+
and MTS24
epithelial cells. From the adult

thymus, MTS24
+
cells can be retrieved, but
their functionremains tobe determined(135).
If MTS24 is perhaps not the TEC pro-
genitor marker, is there any evidence for stem-
ness among TEC? Interestingly, TECexpress
genes suchas Nanog, Oct4, and Sox2, whichare
commonly found in stem cells, and the ex-
pression of these hallmark genes is reduced
in 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 be
important to determine the frequencies of
TEC and their maturation stages that express
Nanog, Oct4, and Sox2. Perhaps TEC stem or
progenitors will be found within these cells or
among cells expressing p63 (136) (see below).
GENES AFFECTING THYMUS
DEVELOPMENT AFTER
THYMUS SPECIFICATION
Foxn1, the Gene Mutated
in the Nude Mouse
There is no denitive marker that indicates
epithelial cell commitment toward thymus
fate prior to expression of Foxn1. Function-
ally speaking, Foxn1 is the single most impor-
tant gene known to be essential specically
for thymus epithelial development (60, 61).
Therefore, it is worthwhile to take a closer
look at properties of Foxn1 such as expres-
sion, regulation, and function. Foxn1 is a pro-
tein belonging to the family of forkhead box
transcription factors (137139). In addition to
Foxn1, 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, and
Foxo factors, which are involved in apoptosis
and proliferation, and hence leukocyte home-
ostasis (138, 139). Foxn1 is characterized by
a winged-helix/forkhead DNA-binding do-
main and a transcriptional activation domain
(42, 140). In the original nude mouse allele
(Foxn1
nu
), a single base pair deletion in exon
3 causes a frame shift leading to a truncated
Foxn1 protein lacking both the DNA-binding
and the activation domain (60). Homozygous
Foxn1
nu/nu
mice had the same thymus and skin
phenotypes as compound heterozygous mice
bearing one engineered null allele of Foxn1
(Foxn1
) and one natural mutant allele of

Foxn1 (Foxn1
nu
), formally showing that Foxn1
is allelic to the nude gene (61).
In mice homozygous for a Foxn1 allele
lacking exon 3 (Foxn1
/
), thymus epithe-
liumdevelopedbeyondthe block inFoxn1
nu/nu
mice. Foxn1
encodes a Foxn1 protein with a

large deletion in the N-terminal part of Foxn1
(141). The phenotype of Foxn1
/
mice over-
all resembled that of a hypomorphic mutant in
which TEC phenotype and organization sug-
gest an arrest at a K5
+
K8
+
stage. In contrast
to the cystic Foxn1
nu/nu
thymus, the Foxn1
/
thymus had a more continuous structure per-
missive to support T cell development, albeit
only poorly andwitha delay infetal thymocyte
development. It is not clear whether this TEC
phenotype results froma specic requirement
for the N-terminally deleted amino acids at
later stages of TEC development, perhaps
beyond the K5
+
K8
+
stage, or whether the
Foxn1
allele encodes a Foxn1 protein with an

overall reduced functional activity. The sta-
bility of the Foxn1 protein encoded by this
particular allele has not been determined.
Little is known about upstreamfactors that
regulate Foxn1 expression. Through trans-
genic work, DNAfragments and promoter el-
ements that permit gene expression under the
control of Foxn1 have been described (41, 93,
142). It has been proposed that Foxn1 expres-
sion is regulated through members of the Wnt
372 Rodewald
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family (143). Given that no single or com-
pound Wnt mutant mouse has been reported
that resembles a nude thymus phenotype, fur-
ther clarication of the role of Wnt family
members directly upstreamof Foxn1 would be
helpful. In brief, there is very little informa-
tion on how Foxn1 transcription is initiated
in development or maintained in the adult
thymus.
On the basis of a Foxn1
lacZ
knockin allele,
in situ hybridization, and antibody staining,
investigators have found that Foxn1 expres-
sion in thymic epithelium is rst detectable
on E11.5 (6163), a stage preceding coloniza-
tion of the thymus by hematopoietic progen-
itors. Analysis of a Foxn1
lacZ
allele demon-
strates that Foxn1 is expressed in most, if not
all, thymic epithelial cells both at embryonic
stages and in the adult (61). However, a more
recent study that used an anti-Foxn1 anti-
body suggests that both Foxn1
+
keratin
+
and
Foxn1
keratin
+
thymic epithelial cells exist
in the embryonic thymus (E13) and that the
percentage of Foxn1
keratin
+
TECis even as
high as 80% in the adult thymus (63). If sub-
sets of adult TEC differ, in fact, in expression
of Foxn1, it would be interesting to dene how
those Foxn1
+
and Foxn1
subsets differ with

regardtothe relative stages of maturation, cel-
lular age, turnover, or their functional capaci-
ties to promote Tcell development. Although
the extent towhichadult TECactively express
Foxn1 is controversial, fate mapping of TEC
using a Foxn1
Cre
allele convincingly showed
that most, if not all, TEC arise from Foxn1
+
progenitors, or at least transit through a stage
of ubiquitous Foxn1 expression (39, 40). As
mentioned earlier, genetic activation of Foxn1
in single TEC led to the appearance of units
of productive thymi in an otherwise nude thy-
mus. This underscores the idea that Foxn1
is expressed in thymus-forming progenitors
(26).
Foxn1-dependent cells in the thymus can
be visualized in nude blastocyst comple-
mentation (Figure 2) (9) by injection of
Gfp
+
Foxn1
+
ES cells into Gfp
Foxn1
nu/nu
blastocysts (Figure 5). On thymus tissue sec-
a
b
c
DAPI = all nuclei
Gfp= Foxn1-dependent TEC
ES cells
Gfp
+
Foxn1
+/ +
Blastocysts
Gfp
Foxn1
nu/nu
Estimation of number and
location of Foxn1-dependent
TEC by visualization of
Gfp
+
nuclei
Figure 5
Visualization of Foxn1-dependent thymus epithelium. The rare
representation of TEC among all thymus cells is demonstrated in chimeric
mice generated by injection of Gfp
+
Foxn1
+/+
embryonic stem (ES) cells
(a) into Gfp
Foxn1
nu/nu
blastocysts (b). Percentages, phenotypes and tissue
distribution 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
(<5%) ES cell contribution to thymocytes or thymic mesenchyme, and
hence the Gfp
+
cells represent mostly Foxn1-dependent thymus
epithelium. Note the abundance of mTEC (inner area) and, by comparison,
the paucity of cTEC (outer area), which implies that the density of the two
TEC types is different.
tions, Gfp expression is conned to the cells
nucleus by nuclear localization and indicates
the position of cells from ES cell origin. In
the example shown in Figure 5, there was
minimal contribution (<5%) of the ES cell
to hematopoietic cells (thymocytes) and to
nonepithelial stromal cells, and hence the vast
majority of Gfp
+
cells in the thymus repre-
sent cells that are dependent in development
or maintenance on Foxn1 expression, that is,
they are TEC. Overall, the density of these
Foxn1-dependent TEC is much higher in the
medulla than in the cortex. This is perhaps
surprising given that the cortex space is com-
pletely lled with keratin
+
cells, but the dis-
tribution of Gfp
+
cells indicates that mTEC
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and cTEC appear to cover different thymus
volumes per cell. The conclusion that mTEC
outnumber cTEC in situ is certainly compat-
ible with the observation made by many labo-
ratories that proportionally more mTECthan
cTEC are routinely retrieved from stromal
cell preparations.
Lack of Foxn1 expression in nude mice
becomes phenotypically evident as early as
E12.5 or 13.5 when the Foxn1-decient thy-
mus anlage fails to grow adequately when
compared to wild-type thymus (22, 42, 144,
145). The nude thymus rudiment is also char-
acterized by a near absence of hematopoietic
cells (22, 42, 146), possibly related to loss of
expression of the chemokines CCL25 (ligand
of CCR9) and CXCL12 (ligand of CXCR4)
inthe embryonic nude thymus (147). Interest-
ingly, embryonic nude thymic epithelial cells
are also decient in expression of the Notch
ligands Dll1 and Dll4 (145). Although the
question whether Notch-Notch ligand inter-
actions and concomitant T cell commitment
take 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 nude
thymus may be prohibitive for T cell devel-
opment and, as such, may contribute to the
alymphoid nude thymus phenotype.
Virtually all medullary and cortical TEC
in chimeras constructed from Foxn1
+/+
plus
Foxn1
nu/nu
embryos (149, 150) or from
Foxn1
+/+
ES cells plus Foxn1
nu/nu
blastocysts
(9) were fromthe Foxn1
+
but not Foxn1
nu
ori-
gin. This demonstrates that Foxn1 acts in a
cell-autonomous manner in thymic epithelial
cells. The action of the Foxn1 gene in cis, with
no rescue in trans, implies that the essential
target genes of the Foxn1 transcription fac-
tor do not include a gene encoding a soluble
factor such as a growth factor or a cytokine,
or at least none that plays a substantial role in
the nude thymus phenotype. Although genes
have been identied that are differentially ex-
pressed between Foxn1
+
and Foxn1
nu
TECs
(144), functionally crucial target genes are
unknown. This may appear surprising given
that Foxn1 has been known for some time. It
should be noted, however, that it has been im-
possible tostudy the true functionof Foxn1ei-
ther inthe development or inthe maintenance
of thymus epithelium in cell lines in vitro.
This necessitates analyses of primary thymic
epithelial cells that are rare cells (on the or-
der of a few percent of total thymus cells) in
a normal thymus and are even rarer in a nude
thymus. 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 biochemical
studies, including the identication of rele-
vant DNA binding sites or protein cofactors
that regulate the transcription factor activity
of Foxn1 in primary TEC. As a consequence,
the molecular function of Foxn1 in thymic ep-
ithelium remains enigmatic. Likewise, it will
be of interest to determine the role of Foxn1
beyond the developmental block in nude
mice, that is, in differentiated thymus epithe-
lium and perhaps during thymus involution.
Traf6 and RelB
TEC differentiation is completely blocked in
Foxn1
nu/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 the
TNFreceptor-associatedfactor Traf6. Never-
theless, both of these mutants show major de-
fects in TEC structure and function that may
indicate faulty TECdifferentiationor mainte-
nance. Traf6 is a signal transducer in the NF-
B pathway that activates IB kinase (IKK) in
response to proinammatory cytokines. IKK
converts 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 is
used in the developing thymus in cells with
a medullary location (152). In the absence of
Traf6 (153), the thymus architecture is disor-
ganized and the medulla-cortex separation is
blurry. Medullary TECpopulations are either
missing or lack expression of certain cell sur-
face markers (UEA-1
but K5
+
phenotype).
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These mutants suffer fromautoimmunity that
correlates strongly with lack of Aire
+
TEC,
concomitantly reduced expression of tissue-
restricted self-antigens on the TEC side, and
an absence of Foxp3
+
regulatory T cells in
the thymus. These defects are indeed stromal
cellintrinsic, as shown by transfer of autoim-
mune symptoms by thymus grafting. RelB
expression was markedly reduced in thymus
stroma ex vivo and in TECcell lines and could
be restored by reintroduction of Traf6. The
diminished expression of RelB is a plausible
molecular mechanism because earlier work
has shown that RelB-decient mice also have
severe medullary defects that render mice au-
toimmune (154156). The phenotype of the
Traf6-decient thymus has been proposed to
be independent of LTR ligand signaling on
mTEC (118) because LTR-induced activa-
tion of NF-B was unaffected by lack of Traf6
(153). Overall, the importance of different
NF-B pathways in thymus epithelium is not
entirely clear because Traf6-independent NF-
B activity was noted in the aforementioned
reporter mouse (152).
The molecular defects in mice lacking RelB
or Traf6 give novel insights into the genetic
requirements for a key thymus function, tol-
erance induction. However, further interpre-
tations of these and other studies into TEC
defects are complicated by the ambiguity of
whether the gene defects cause ablation of a
cell type (e.g., Aire-expressing mTEC) or al-
ter the gene expression (e.g., Aire in otherwise
normal TEC).
p63
Expression of p63, a homolog of the tumor
suppressor p53, is required for the normal de-
velopment and maintenance of many epithe-
lial tissues. The widespread epithelial defects
in mice lacking p63 are consistent with a cru-
cial function of this gene in epithelial stem
cells. p63
/
mice possess very small thymi
that were recently analyzed in detail (136,
157). p63 was expressed in all K8
+
thymic
epithelial cells on E12 and continues to be
expressed later in ontogeny in some but not
all medullary and cortical TEC. Numbers of
thymocytes were very low, but T cell devel-
opment was normal in p63
/
thymi. Hence,
mutant TECare, inprinciple, functional (136,
157). This also holds true for hematopoi-
etic progenitors, as shown by T cell devel-
opment from p63
/
fetal liver stem cells
transferred into Rag-2-decient mice. Fur-
ther experiments showthat TECfromp63
/
thymi have reduced expansion potential in a
general 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 thymus
epithelium that was considered a thymus ep-
ithelial stem cell assay. Cells that expanded
in this assay expressed p63, and knock-down
of p63 strongly impaired the colony size, di-
rectly supporting the argument for an impor-
tant role of p63 in TEC or TEC stem cell
maintenance.
Because support of T cell development
is the ultimate functional test for TEC, it
would be interesting to know whether TEC
that 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, some
of which are familiar as they play a role in
thymus organogenesis. Notably, FgfR2-IIIb,
which was mentioned earlier as a crucial re-
ceptor involved inmesenchymal-epithelial in-
teractions (72), as well as Jag2were downregu-
lated (157). Collectively, these recent ndings
on the role of p63 in TEC development, and
possibly TEC stem cell maintenance, may
open new experimental access to the iden-
tity of thymus stem cells and their molecular
regulation.
FUNCTIONAL CERVICAL
THYMUS IN MICE
Incidence
Recently, evidence has been provided that
mice have a cervical thymus that is indeed
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functional in its ability to be colonized by
bone marrowderived progenitors, to gener-
ate thymocytes that are TCR repertoire se-
lected according to the laws of positive and
negative selection, and to export mature im-
munocompetent Tcells. Terszowski et al. (41)
found cervical thymus in chimeras generated
as controls for mutant chimeras initially made
to test the function of Tbx1 in thymus organo-
genesis. Dooley and colleagues (158) searched
for ectopic thymus in the neck based on the
similarity of thymus epithelium with non-
thymic epithelium such as respiratory epithe-
lium (90) and on the observation that sam-
ples of human parathyroid showed inclusions
of 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 the
lower but still signicant frequency of 30%
Foxn1:: Egfp
a b
C57BL/6
3 mm 2.6 mm
Neck
Neck
Chest
Chest
Figure 6
Functional cervical thymi in mice. (a) Visualization of bilateral cervical
(neck) thymi in a Foxn1::Egfp reporter mouse, and (b) histological
comparison of May-Gr unwald-Giemsa-stained tissue sections from the
thoracic thymus and one cervical thymus lobe in a C57BL/6 mouse. The
functional properties of neck thymus have recently beendescribed(41, 158).
(158) to 50% (41) in C57BL/6 mice. These
differences might be explained by the differ-
ent counting of either all detectable (41) or
only medially located thymi (158). As other
tools 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 shown
in a mouse line (FVB C56BL/6 back-
cross) transgenic for a Foxn1
Egfp
reporter gene
in which Foxn1-expressing cells are visual-
ized specically by green uorescence (41)
(Figure 6a), and in a normal C57BL/6 mouse
inwhichtwothoracic andone cervical lobe are
displayed as May-Gr unwald-Giemsa-stained
tissue sections (Figure 6b). Hence, although
cervical thymi are not detectable in every sin-
gle mouse in the strains analyzed so far, two
of the most commonly used laboratory strains
(BALB/c and C57BL/6) frequently have cer-
vical thymi. Although the neck region of nude
mice has, to my knowledge, not formally been
examined for cervical thymi, the known lack
of functional Tcells in this mutant and the ex-
pression of Foxn1 in TEC in the neck thymus
(41, 158) support the argument for a common
defect in the generation of thoracic and cer-
vical thymi in the nude mouse.
Thoracic and Cervical Thymus
in Other Mammals
The cervical thymus in mice is not with-
out precedent, because neck thymus is known
from other mammals. Primitive mammals
such as marsupials (pouched mammals) pro-
vide interesting examples. Most marsupials
have 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. In
sheep, cattle, and pigs, the thymus has cervical
and thoracic parts belonging to one thymus
andtherefore they are connectedtoeachother
(160). In humans, case reports of disease-
associated cervical thymi (161163) suggest
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that cervical thymus is rare (see references in
41, 158) and is caused by failure of the thy-
mus to properly descend to its nal mediasti-
nal location. However, others have estimated
that the cervical thymus in humans may be
quite frequent, with an incidence of 50% in
adults (further references in 158) and 60% in
children (164). Collectively, precise gures on
the normal incidence of neck thymus in hu-
man are difcult to extract from the literature
(for further references and considerations, see
also 162), and a denitive answer to this ques-
tion may require further and more systematic
imaging data.
Origin of the Cervical Thymus
in Mice and the Relationship
of Thymus and Parathyroid
The following possibilities can be considered
for the origin of the second thymus.
1. The cervical thymus originates fromthe
thirdpouchas the thoracic thymus does,
but it takes the parathyroid route of mi-
gration. This could occur as a result
of imprecise separation of the parathy-
roid and thymus domains (62) within
the third pouch epithelium, leading to
the partition of thymus progenitors into
the 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 parathyroid
organs do not only develop from the
same pharyngeal pouch but also share,
at least in mice, some functional sim-
ilarities. Some 70% of Gcm2-decient
mice survive despite a complete absence
of parathyroids (64). In mice, but not in
humans (165), the thymus was identied
as the auxiliary source of parathyroid
hormone (PTH) (64). However, though
expression of Gcm2 is restricted to the
parathyroid and is required for the de-
velopment of precursors for this organ
(104), the PTH-producing cells in the
thymus colocalize with Gcm1, a gene
homologous to Gcm2, that is expressed
in clusters of cells in the thymus (166).
The lineage of these Gcm1-expressing
cells in the thymus is unknown.
2. The anlage for the cervical thymus
arises independently in a different
pouch, and, up to now, these few TEC
progenitors may have escaped detec-
tion. An origin in a separate pharyn-
geal pouchwouldbe akintothe multiple
thymus anlagen in the shark (Figure 3)
and could be considered an atavism.
3. There could be a delayed epithelial
specicationtoward the cervical thymus
fate 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 provide
a clue. This latter idea is supported by
the notion that the development of the
cervical thymus is delayed by about one
week compared to the thoracic thymus
(41) (see below).
Developmental Kinetics
of the Cervical Thymus
The identication of the primitive anlage for
the cervical thymus in the neck of newborn
mice also led to the surprising result that tho-
racic and cervical thymi do not develop in par-
allel but asynchronously (41). Cells in the cer-
vical thymus anlage in the neck of newborn
mice express Foxn1 and MHCclass II, indica-
tive of their thymus identity, but, at this stage,
the overall TEC structure is still primitive,
with many cells coexpressing K5 and K18 and
poor epithelial cell compartmentalization. In
addition, there are only few and only imma-
ture thymocytes present in the neck thymus
at this stage (41). TCR DJ and V(D)J re-
arrangements are undetectable in the neck
on fetal day 18.5, further supporting the lack
of thymic function in the neck before birth.
Based on the kinetics and a maturational com-
parison of cervical and thoracic thymus, the
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cervical thymus appears at birth as immature
as the major thymus one week earlier, that is,
on days 12 or 13 in embryonic development.
The developmental delay between the tho-
racic and the cervical thymus is also evident by
a burst of CD4
+
CD8
+
cells as late as one week
after birth in the neck thymus, as opposed to
the same burst, on a larger scale, in the thorax
thymus on fetal day 18. The presumed onset
of thymopoiesis in the neck only after birth
could make the cervical thymus miss the wave
of production of V5-expressing thymocytes
(G. Terszowski & H.-R. Rodewald, unpub-
lished) that are the precursors of the canon-
ical TCR-expressing dendritic epidermal T
cells (167). Along these lines, asynchronous
functioning of thoracic and cervical thymus
within individual mice may give clues as to
which properties of the thymus are organ-
autonomous and which are regulated at the
level of the organism or at the level of the
bone marrowthat is producingthymus-bound
progenitors.
Anatomy
Cervical thymi in the mouse are often single
unilateral lobes, but two bilateral (Figure 6)
or even two unilateral lobes can also be found.
Cervical thymi are usually located medially,
that is, on the inside of the large cervical ves-
sels, and along the ventral region between the
thyroidor evenmore cranially towardthe sub-
mandibular glands and caudally toward the
lower part of the neck. However, in no in-
stance could we nd open connections to the
chest or a continuous chest-neck-thymus in
mice. The position of the cervical thymus is
usually supercial, but cervical thymus lobes
are also present between or under muscle
strings (for anatomical positions, see 41, 158).
Overall, the distribution of cervical thymus is
much wider than previously suggested on the
basis of previous anatomical reports of thymus
tissue located within or next to the thyroid
(168, 169). Likewise, the view that mice, sim-
ilar to sheep or cattle, have a cervical part of
their thymus (170) is not supported by recent
studies (41, 158). Many of these discrepancies,
and the fact that cervical thymus in mice has
by and large been overlooked in immunology,
could be explained by the fact that cervical
thymus lobes may have been considered cer-
vical lymph nodes. Cervical thymi can easily
be distinguished from lymph nodes by histol-
ogy or ow cytometry using thymus-specic
markers (41, 158).
Function of the Cervical Thymus
and Implications
Several differences have been noted compar-
ing cervical and thoracic thymus. Cervical
thymi are mostly single small lobes composed
of medulla and cortex, whereas thoracic thymi
are, of course, composed of multiple medulla
and cortex structures (41, 158) (Figure 6).
Thymic broblasts are found more locally re-
stricted in the neck than the chest thymus
(158). The cortex in the cervical thymus con-
tains clusters of CD4
+
CD8
cells that are

unusual among normal cortical CD4
+
CD8
+
cells (41). The cervical thymus has between
10
5
and 2 10
5
cells but these cell numbers
can also be smaller or be as large as 10
6
.
This cellularity is low when compared to the
thoracic thymus (10
8
cells) but large when
compared to, say, single gut cryptopatches,
intestinal areas harboring 10
3
lymphoid
progenitors (171, 172).
When tested in model systems for positive
and negative TCR repertoire selection, tho-
racic and cervical thymus obey the same well-
known rules (41, 158). Moreover, the cervical
thymus can export functional T cells when
grafted into nude mice (41, 158), and these
T cells mount T cell help required for an
antibody response, including class switching
(41). The contribution of the cervical thymi
to the overall T cell pool in a mouse that
has both thoracic and cervical thymi is un-
known, and it may be proportional to the
size differences. Likewise, the contribution
of cervical thymi to peripheral T cells in a
378 Rodewald
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mouse that underwent thoracic thymectomy
remains to be determined. The cervical thy-
mus contains Foxp3
+
thymocytes intheir nor-
mal medullary location, suggesting that the
neck thymus provides both effector and reg-
ulatory T cells to the periphery (158). This
raises some interesting questions regarding
the possible role of the cervical thymus in
models of autoimmunity that are driven by
thoracic thymectomy shortly after birth (173).
Because cervical thymi have not beenremoved
in these experiments, it remains to be deter-
mined whether T cells produced by the cervi-
cal thymus postnatally contribute to this phe-
nomenon or not, and if so, why they cannot
be controlled by regulatory cells that can also
be generated in the cervical thymus.
Hints that mice have a thymus intheir neck
date back to 1964. A histological section from
the parathyroid region of a BALB/c mouse
neck included thymus tissue (168; discussed
in 174, 175). Moreover, cervical thymus em-
bedded in the thyroid gland in 80% of female
diabetes-prone NOD but not in normal con-
trol mice had been reported (169). Cervical
thymi in lieu of normal thoracic thymi were
identied in Pax9-decient mice, providing a
case for ectopic thymus as a result of failed
migration toward the thorax (87). Anatomical
drawings, on the other hand, suggested that
mice, similar to sheep (160), have a cervical
part of the thymus (170), a viewnot supported
by the recent analyses (41, 158). In any case,
the function of the cervical thymus in mice
was clearly unknown. Moreover, as far as we
know, neither the proper anatomical location
(as opposed to cervical lymph nodes) nor the
incidence had been reported.
It is clear now that the cervical thymus,
though not detectable in every individual
mouse, is not restricted to the BALB/c strain,
and it is also not restricted anatomically to
the thyroid/parathyroid area, nor is it part of
the thoracic thymus. Arguments surrounding
early hints of a cervical thymus, and the fact
that it had been ignored in subsequent experi-
mentation, have recently been exchanged (for
a discussion and further references, see 174
176). Because the cervical thymus is capable
of generating and releasing functional T cells
that render athymic mice immunocompetent,
at least toward antigens tested so far, it may
have confounded thoracic thymectomy exper-
iments. Determining to what degree this is the
case requires further work. Finally, compar-
ative analyses of thoracic and cervical thymi
may yield insights into the regulation of thy-
mus organogenesis, the location dependency
of thymus functions, and possibly thymus
aging.
DISCLOSURE STATEMENT
The author is not aware of any biases that might be perceived as affecting the objectivity of
this review.
ACKNOWLEDGMENTS
I thank past and present members of my laboratory, in particular Carmen Blum, Verena
B uhrmann, Susanna M uller, Sabine Paul, Greg Terszowski (Ulm), and Corinne Haller (Basel),
for their contributions to our studies addressing thymus development. I amgrateful to Graham
Anderson, Thomas Boehm, Louis Du Pasquier, Hans J org Fehling, Reinhard Pabst, and Nick
Trede for advice and discussions, Louis Du Pasquier for drawings incorporated into Figure 3,
Virginia Papaioannou for providing Gfp-marked ES cells (Figure 5), and Thomas Boehm for
collaborating on the Foxn1::Egfp reporter mouse (Figure 6). Thymus research in my labo-
ratory is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 497-B5 and
Klinische Forschergruppe 142-P8).
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AR338-FM ARI 12 January 2008 18:11
Annual Review of
Immunology
Volume 26, 2008
Contents
Frontispiece
K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x
Doing What I Like
K. Frank Austen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Protein Tyrosine Phosphatases in Autoimmunity
Torkel Vang, Ana V. Miletic, Yutaka Arimura, Lutz Tautz, Robert C. Rickert,
and Tomas Mustelin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29
Interleukin-21: Basic Biology and Implications for Cancer
and Autoimmunity
Rosanne Spolski and Warren J. Leonard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 57
Forward Genetic Dissection of Immunity to Infection in the Mouse
S.M. Vidal, D. Malo, J.-F. Marquis, and P. Gros p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 81
Regulation and Functions of Blimp-1 in T and B Lymphocytes
Gisline Martins and Kathryn Calame p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133
Evolutionarily Conserved Amino Acids That Control TCR-MHC
Interaction
Philippa Marrack, James P. Scott-Browne, Shaodong Dai, Laurent Gapin,
and John W. Kappler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 171
T Cell Trafcking in Allergic Asthma: The Ins and Outs
Benjamin D. Medoff, Seddon Y. Thomas, and Andrew D. Luster p p p p p p p p p p p p p p p p p p p p p 205
The Actin Cytoskeleton in T Cell Activation
Janis K. Burkhardt, Esteban Carrizosa, and Meredith H. Shaffer p p p p p p p p p p p p p p p p p p p p 233
Mechanism and Regulation of Class Switch Recombination
Janet Stavnezer, Jeroen E.J. Guikema, and Carol E. Schrader p p p p p p p p p p p p p p p p p p p p p p p 261
Migration of Dendritic Cell Subsets and their Precursors
Gwendalyn J. Randolph, Jordi Ochando, and Santiago Partida-Snchez p p p p p p p p p p p p p 293
v
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AR338-FM ARI 12 January 2008 18:11
The APOBEC3 Cytidine Deaminases: An Innate Defensive Network
Opposing Exogenous Retroviruses and Endogenous Retroelements
Ya-Lin Chiu and Warner C. Greene p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 317
Thymus Organogenesis
Hans-Reimer Rodewald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 355
Death by a Thousand Cuts: Granzyme Pathways of Programmed Cell
Death
Dipanjan Chowdhury and Judy Lieberman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 389
Monocyte-Mediated Defense Against Microbial Pathogens
Natalya V. Serbina, Ting Jia, Tobias M. Hohl, and Eric G. Pamer p p p p p p p p p p p p p p p p p p p 421
The Biology of Interleukin-2
Thomas R. Malek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 453
The Biochemistry of Somatic Hypermutation
Jonathan U. Peled, Fei Li Kuang, Maria D. Iglesias-Ussel, Sergio Roa,
Susan L. Kalis, Myron F. Goodman, and Matthew D. Scharff p p p p p p p p p p p p p p p p p p p p p 481
Anti-Inammatory Actions of Intravenous Immunoglobulin
Falk Nimmerjahn and Jeffrey V. Ravetch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 513
The IRF Family Transcription Factors in Immunity and Oncogenesis
Tomohiko Tamura, Hideyuki Yanai, David Savitsky, and Tadatsugu Taniguchi p p p p p 535
Choreography of Cell Motility and Interaction Dynamics Imaged
by Two-Photon Microscopy in Lymphoid Organs
Michael D. Cahalan and Ian Parker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 585
Development of Secondary Lymphoid Organs
Troy D. Randall, Damian M. Carragher, and Javier Rangel-Moreno p p p p p p p p p p p p p p p p 627
Immunity to Citrullinated Proteins in Rheumatoid Arthritis
Lars Klareskog, Johan Rnnelid, Karin Lundberg, Leonid Padyukov,
and Lars Alfredsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 651
PD-1 and Its Ligands in Tolerance and Immunity
Mary E. Keir, Manish J. Butte, Gordon J. Freeman, and Arlene H. Sharpe p p p p p p p p 677
The Master Switch: The Role of Mast Cells in Autoimmunity
and Tolerance
Blayne A. Sayed, Alison Christy, Mary R. Quirion, and Melissa A. Brown p p p p p p p p p p 705
T Follicular Helper (T
FH
) Cells in Normal and Dysregulated Immune
Responses
Cecile King, Stuart G. Tangye, and Charles R. Mackay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 741
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ANRV338-IY26-12 ARI 16 February 2008 12:33

cells are a mixture of mes-

cellsby default collectively con-

cells include two lineages: Thymus epithelial cells (TEC, Keratin

), which are a mixture of

cells is about 50 to 1, but most

mice (thymus stromal phenotypes reviewed

cells gave rise to

TEC (the majority of which are mTEC) in-

cells (12, 13), others reported thy-

cells are nonepithe-

(that is, nonepithelial)

epithelial from day 15.5 thymus

epithelial cells. From the adult

) and one natural mutant allele of

encodes a Foxn1 protein with a

allele encodes a Foxn1 protein with an

subsets differ with

cells that are

inducer cells regulate development of Aire-expressing epithelial cells in

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