PDGFR α-Expressing Mesenchyme Regulates Thymus Growth And The Availability Of Intrathymic Niches

Correspondence should be addressed to G. Anderson Address: MRC Centre for Immune Regulation, Institute for Biomedical Research, Birmingham University Medical School, Birmingham B15 2TT, UK Tel: 0044-121-414 6817 FAX: 0044-121-464-3599 Email: g.anderson@bham.ac.uk Words of abstract: 200 Word of text: 4,025 Scientific heading: HAEMATOPOIESIS Blood First Edition Paper, prepublished online September 28, 2006; DOI 10.1182/blood-2006-05-023143


Introduction
The production of a functionally competent peripheral T-cell pool with a diverse T-cell receptor (TCR) repertoire is essential in mounting an effective immune response to invading pathogens.Most, if not all, peripheral CD4 ϩ and CD8 ϩ T cells bearing the ␣␤ form of the TCR complex are generated in the thymus from migrant progenitors that arise in the fetal liver and bone marrow. 1,2lthough recent evidence shows that these progenitors may show some degree of commitment to the T-cell lineage prior to thymus colonization, [3][4][5] entry into the thymus and contact with the specialized microenvironment provided by thymic stromal cells is required to induce further development along the T lineage.7][8] During these early phases, DN T-cell precursors undergo commitment to the ␣␤ or ␥␦ lineage, and in the case of the former, signaling through the pre-TCR complex is required for maturation beyond the DN3 stage and progression to the CD4 ϩ CD8 ϩ stage, where the ␣␤ TCR is first expressed. 9As somatic recombination of TCR␣ and TCR␤ chain genes occurs randomly, CD4 ϩ CD8 ϩ thymocytes are then screened on the basis of their ␣␤ TCR specificity by positive and negative events before further maturation into the CD4 ϩ and CD8 ϩ lineages. 10Ultimately, these developmental processes result in the formation of self-tolerant major histocompatibility complex (MHC) class I-restricted CD8 ϩ and MHC class II-restricted CD4 ϩ T cells that leave the thymic medulla and contribute to the peripheral T-cell pool. 11,12ere is now accumulating evidence that the epithelial compartment of the thymic stroma plays a crucial role in supporting these developmental processes.This includes the expression of Notch ligands 13,14 that interact with Notch on DN thymocytes to provide essential maturation signals [15][16][17] as well as the production of secreted factors, including Wnts, 18 interleukin-7 (IL-7), 19 and stem cell factor (SCF), 20 that also play key roles during thymocyte development.Moreover, thymic epithelial cells are heterogeneous, consisting of subcapsular, cortical, and medullary subsets that are likely to provide specialized microenvironments to support particular stages of T-cell development. 1,21For example, cortical epithelial cells a play key role in positive selection; the availability of cortical niches have been shown to be a limiting factor in this process. 22,23n the other hand, the medullary epithelium and dendritic cells play specialized roles in negative selection 24,25 and may influence the development of regulatory T cells. 26In this context, developmental abnormalities affecting different thymic epithelial cell compartments have been shown to result in T-cell disorders such as autoimmunity 27,28 and T-cell immunodeficiency, 29,30 emphasizing the crucial role of thymic epithelial cell populations in the development of an extensive peripheral T-cell repertoire that is nonreactive to self antigen.
In this study, we have investigated the mechanisms regulating the development of thymic epithelial cells leading to the formation of functionally competent thymic microenvironments.We show that during normal development, thymus growth involves a temporally regulated phase of thymic epithelial proliferation, resulting in an increase in the number of stromal-cell niches able to support thymocyte development.In addition, we have defined a population of nonepithelial platelet-derived growth factor receptor ␣-positive (PDGFR␣ ϩ ) fetal mesenchymal cells and shown that the presence and developmental kinetics of this specialized subset of stromal cells correlates with the pattern of epithelial proliferation, consistent with an important role for these cells as a source of signals regulating normal thymus growth.We also present in vivo functional evidence that, in the absence of such mesenchyme, the differentiation of thymic epithelial subsets from bipotent progenitors recently shown to be present in the embryonic day (E) 12 thymus rudiment 31 is initiated normally, but the thymus remains hypoplastic.Such hypoplastic thymi support a normal pattern of T-cell development but with greatly reduced overall numbers of thymocytes, reflecting the limited availability of epithelial support or niches for thymocyte development and selection.In summary, our data define a crucial role for thymic mesenchyme in the determining thymus size and capacity for T-cell production by regulating the extent of the epithelial microenvironments essential for the development of T-cell progenitors into functional T cells.

Mice
BALB/c (H-2 d ) mice were bred and maintained under specific pathogenfree (SPF) conditions in the Biomedical Services Unit at the University of Birmingham.The day of vaginal plug detection was designated as day 0.

Analysis of thymic epithelial proliferation
Thymus lobes were dissected from mice of the indicated ages and disaggregated using 0.25% trypsin (Sigma).Suspensions of cells from neonatal thymi were stained with APC-conjugated anti-CD45 to aid in the identification of CD45 Ϫ stroma.Cells were permeabilized using the eBioscience Fixation and Permeabilization kit.FITC-conjugated anti-pancytokeratin and PE-conjugated anti-Ki67 (clone B56; BD Pharmingen) were used to detect thymic epithelial proliferation.

Immunohistochemistry
Frozen sections of thymic grafts, recovered from under the kidney capsule after 3 weeks, were prepared as described. 32Tissue sections of 5 m thickness were fixed in ice-cold acetone, and stained with either anticytokeratin 5 (Covance, Berkeley, CA) and anti-cytokeratin 8 (clone LE41; kind gift from B. Lane, University of Dundee, Scotland) or the panmesenchyme marker ERTR7 (kind gift from W. van Ewijk, Leiden University Medical Centre, the Netherlands).Primary antibodies were detected using anti-rabbit Alexa Fluor 350 (Molecular Probes, Eugene, OR), anti-mouse FITC (Caltag, Burlingham, CA) or anti-rat FITC (Southern Biotech, Birmingham, AL), respectively.Sections were mounted in antifade glycerol solution (Citifluor, Canterbury, United Kingdom).Sections were viewed using a Zeiss Axioplan microscope (Welwyn Garden City, United King-dom).Tissue sections were analyzed using an Axioplan 2 microscope (Zeiss, Jena, Germany) fitted with Zeiss Plan Neofluar objectives (20ϫ/ 0.50 and 40ϫ/1.3).Images were captured using a Hamamatsu Orca-ER camera (Welwyn Garden City, United Kingdom), and analyzed using SmartCapture X software version 2.5.9 (Digital Scientific, Cambridge, United Kingdom).

Analysis of the cellular compartments of the E12 thymus
E12 thymic lobes with surrounding mesenchyme still attached were dissected with the aid of a stereo-dissecting microscope and no. 5 Watchmakers forceps (Taab, Aldermaston, United Kingdom).Freshly isolated lobes were disaggregated by incubation in 0.25% trypsin and then labeled with antibodies to EpCAM1, PDGFR␣, and CD45, as described. 5

Preparation of mesenchyme-free thymic epithelial rudiments for transplantation
To remove the surrounding perithymic mesenchyme from the inner epithelial core, E12 thymus lobes were incubated at 37°C for 20 minutes in 2.5 mg/mL collagenase D (R&D Systems, Minneapolis, MN) in Ca 2ϩ/ Mg 2ϩfree PBS.Under direct visual observation using a dissecting microscope, lobes were then drawn into a fine, mouth-controlled glass pipette to separate surrounding mesenchyme from the inner epithelial rudiment.To control for the possibility that removal of perithymic mesenchyme damages the inner epithelial core, stripped epithelial rudiments were reassociated overnight with E12 perithymic mesenchyme, and then grafted under the kidney capsule as described below, under "Kidney capsule transplantation."In some experiments, mesenchyme-stripped and intact E12 thymus lobes were transferred into fresh solutions of 0.25% trypsin in 0.02% EDTA to produce a single-cell suspension for flow cytometric analysis.

Kidney capsule transplantation
Whole E12 thymus lobes, or lobes with perithymic mesenchyme removed, were placed under the renal capsules of 4-to 6-week-old syngeneic mice, as described. 33After 3 weeks, grafts were harvested and analyzed for thymocyte cellularity and T-cell development, or by immunohistochemistry, as appropriate.

Detection of host-derived mesenchyme in thymus grafts
To identify the presence of host-derived mesenchyme cells in grafted thymus tissue, E12 thymus lobes were isolated from enhanced yellow fluorescent protein (eYFP) transgenic mice as described 31 and transplanted under the kidney capsules of wild-type (WT) syngeneic mice.After 3 weeks, grafts were harvested and digested with trypsin, and stained with anti-CD45 and anti-EpCAM1 together with Mts15 (a kind gift from R. Boyd and D. Gray, Monash University, Melbourne, Australia) or anti-Ly51, antibodies that react with mesenchyme cells, 33,34 allowing the identification of host-derived (eYFP

Semiquantitative reverse transcriptase-PCR analysis
PDGFR␣ ϩ thymic mesenchyme was prepared from disaggregated E12 thymus lobes, as described above, under "Preparation of mesenchyme-free thymic epithelial rudiments for transplantation."For adult kidney capsulederived mesenchyme, the mesenchymal capsule was carefully peeled away from excised 4-to 6-week-old adult kidneys, and small pieces were placed in 6-well tissue-culture plates.After 48 hours, nonadherent cells were removed, and remaining adherent cells were cultured in DMEM containing 10% FCS.To obtain high-purity cDNA, mRNA was purified from cells using Macs One-step cDNA kit (Miltenyi Biotech, Auburn, CA).␤-actin was used as the housekeeping gene for sample normalization, prior to amplifying the target genes of interest.Reactions were conducted in a Peltier Thermal Cycler PTC-200 (MJ Research, Genetic Research Instrumentation, Braintree, Essex, United Kingdom), as described 5 where during cycling, 3 10-L samples were removed from each reaction in cycling intervals; the range of cycle numbers depended on the gene.Polymerase chain reaction (PCR) products were analyzed by ethidium bromide gel electrophoresis and identified by fragment size.Densitometrical analysis was determined using Syngene Gel Documentation Gene Tools software (Cambridge, United Kingdom).Graphs show ratios of mRNA for the genes of interest relative to ␤-actin.Error bars show SEM of the ratios.

Temporal regulation of thymic epithelial proliferation
The early fetal thymus consists of an endodermally derived epithelial outbudding of the third pharyngeal pouch that is encapsulated by condensing neural crest-derived mesenchyme. 5,35At around E12 of gestation, when the thymic rudiment is undergoing the first wave of progenitor colonization, cortical and medullary microenvironments distinctive of the postnatal thymus are yet to form. 5,35,36Epithelial cells within this early rudiment are still immature and include bipotent progenitors for cortical and medullary epithelial subsets. 31To investigate the events leading to the formation of the definitive thymic epithelial microenvironment, we first examined the proliferative status of cells within the epithelial component of the thymus at successive stages of ontogeny.Flow cytometric analysis was carried out on disaggregated thymus cell suspensions using colabeling with the panepithelial marker cytokeratin and the proliferation marker Ki67 37 to allow quantitative analysis of proliferation specifically within the epithelial component of the thymus (Figure 1A).Using this approach, the proportion of epithelial cells in cell cycle was found to vary during thymus ontogeny, with peak proliferation of cytokeratin ϩ cells observed at E14, followed by a gradual decrease until only a small proportion of epithelial cells were Ki67 ϩ in the neonatal thymus (Figure 1B).Thus, while the early fetal thymus contains a high proportion of proliferating epithelial cells, this decreases with increasing developmental age.

Declining thymic epithelial proliferation correlates with a loss of thymic PDGFR␣ ؉ mesenchyme
To investigate possible mechanisms regulating this temporal pattern of epithelial cell proliferation in the developing thymus, we next analyzed the presence of other cell types that could influence this process.Flow cytometric analysis of disaggregated preparations of E12 thymic rudiments shows that few CD45 ϩ progenitors are present in the thymus at this early stage (Figure 2A), consistent with our previous observations arguing against a role for hemopoietic cells in providing signals regulating initial epithelial proliferation in the fetal thymus. 38,39Further analysis of the E12 thymus found it to consist predominantly of 2 CD45 Ϫ stromal cell types: cytokeratin ϩ epithelial cells, and cytokeratin Ϫ cells, with most of the latter staining positive for expression of the neural crestderived mesenchymal marker PDGFR␣ (Figure 2B).Notably, ontogenetic analysis of this PDGFR␣ ϩ population of mesenchyme in the thymus revealed a temporal reduction in its contribution to the nonhematopoietic nonepithelial component of the thymus (Figure 1B), which correlated closely with the reduction in thymic epithelial proliferation described in the previous section.While the above data show that PDGFR␣ can be used as a useful marker to identify mesenchymal cells, it is not clear whether PDGFR␣ expression is functionally significant, although it is interesting to note that patched mice, which carry a natural mutation in pdgfra, have a smaller thymus compared with that of littermate controls. 40hatever the case, the relationship described here between the presence of PDGFR␣ ϩ mesenchyme and thymic epithelial proliferation is strongly suggestive of a role for these cells in the development of thymic epithelial microenvironments to provide increased numbers of niches for developing thymocytes.

PDGFR␣ ؉ thymic mesenchyme regulates growth but not differentiation of thymic epithelial microenvironments
To test the hypothesis that PDGFR␣ ϩ fetal thymus mesenchyme plays a specific role in regulating thymic epithelial cell development in vivo, we prepared mesenchyme-free thymus rudiments by removing the surrounding perithymic mesenchyme from isolated E12 thymus lobes.Thus, E12 thymus lobes were treated briefly with collagenase to loosen the surrounding mesenchyme, which was then completely removed by drawing the lobes up into a mouth-controlled fine capillary pipette (Figure 3A).Such an approach results in the separation of surrounding mesenchyme, leaving an intact core of thymic epithelium (Figure 3B).Importantly, when epithelial cores prepared in this way were further disaggregated to form a single-cell suspension, in contrast to whole-thymus preparations (Figure 3C), they were found to be devoid of PDGFR␣ ϩ mesenchyme (Figure 3D), indicating the efficiency of the separation to isolate mesenchyme-free thymic epithelial rudiments.
To directly investigate the involvement of PDGFR␣ ϩ fetal thymic mesenchyme in thymus development and growth under in vivo conditions, we grafted both intact and mesenchyme-free E12 thymus lobes under the kidney capsules of adult syngeneic recipients.At this developmental stage, epithelial cells are immature and largely of a keratin 5 ϩ 8 ϩ double-positive phenotype. 38,41hen grafts were harvested after 3 weeks, as in unmanipulated grafts (not shown), epithelial cells within mesenchyme-stripped thymi were found to have differentiated into distinct keratin 5 Ϫ 8 ϩ cortical and keratin 5 ϩ 8 Ϫ medullary subsets together with the appearance of organized cortical and medullary areas (Figure 4A).Moreover, both intact and mesenchyme-stripped grafted thymi were able to support the maturation of T-cell precursors into CD4 ϩ CD8 ϩ and more mature CD4 ϩ CD8 Ϫ and CD4 Ϫ CD8 ϩ subsets expressing the ␣␤ TCR complex (Figure 4B-E).Thus, the ability to form organized cortical and medullary microenvironments that support the development of T-cell precursors indicates that the presence of fetal PDGFR␣ ϩ mesenchyme cells are not required for differentiation of immature thymic epithelial cells.Critically, however, mesenchyme-depleted thymic epithelial rudiments were hypoplastic with a marked absence of growth compared with lobes containing fetal mesenchyme (Figure 5A-B), with hypoplasia being associated with a profound reduction in total thymocyte numbers (Figure 5C).The observed lack of growth of mesenchymestripped thymic rudiments was not due to technical issues caused by the enzymatic separation procedure, as stripped lobes reassociated with fetal mesenchyme grew effectively in vivo and upon harvesting were found to have a thymocyte cellularity comparable with that of unmanipulated grafts (Figure 5C).Furthermore, this lack of thymus growth was not due to a lack of mesenchyme per se within the grafted tissue, as immunohistochemical analysis using the panfibroblast marker ERTR7 showed that mesenchymal cells were readily detectable within stripped thymus lobes (Figure 5D-E).To provide direct evidence for the presence of host-derived mesenchyme in thymus grafts, we transplanted eYFP transgenic E12 thymus lobes under the kidney capsules of WT adult mice.When used in association with anti-CD45 and anti-EpCAM1 to exclude haemopoeitic and epithelial cells, analysis with 2 antibodies shown to react with mesenchyme cells (MTS15 33 and Ly51 34 ) showed the presence of host-derived (eYFP Ϫ ) mesenchyme within the graft (Figure 6).Collectively, these findings suggest that there is a specific requirement for PDGFR␣ ϩ fetal thymic mesenchyme in the expansion of fetal thymic epithelial cells in order to provide sufficient stromal niches for increasing numbers of developing thymocytes.
The presence of adult host-derived mesenchyme in thymus grafts suggests that these cells are unable to provide the appropriate signals that regulate proliferation of E12 thymic epithelial progenitors, making them distinct from fetal PDGFR␣ ϩ mesenchyme cells.To try to identify possible regulators of epithelial progenitor proliferation, we used reverse transcriptase (RT)-PCR analysis to analyze gene expression in adult kidney-derived mesenchyme and E12 PDGFR␣ ϩ thymic mesenchyme.Interestingly, FGF7 and FGF10 mRNAs were detectable in kidney capsule mesenchyme, but not in E12 PDGFR␣ ϩ thymic mesenchyme (Figure 7), suggesting that the lack of growth of E12 thymic epithelium is not due to the lack of availability of FGF7 and FGF10.Such an observation is significant, as we have shown previously that mesenchyme cells present in the E14 thymus provide fibroblast growth factors (FGFs) that regulate epithelial cell proliferation at this later stage.The lack of detectable expression of FGF7 and FGF10 by E12 mesenchyme suggests that a different mechanism regulates the proliferation of bipotent epithelial progenitors present in the E12 thymus. 31Indeed, when we next analyzed expression of IGF1 and IGF2, factors known to be mitogenic for epithelial cells in several tissues, 42,43 we found them to be readily detectable in E12 PDGFR␣ ϩ thymic mesenchyme but not in kidney capsule mesenchyme.Moreover, IGF1 and IGF2 expression by E12 thymic mesenchyme was found to correlate with expression of the IGF1 receptor by E12 thymic epithelial cells, suggesting a role for IGF-IGF1R in early thymus growth.

Discussion
Thymic cortical and medullary epithelial cells provide specialized microenvironments to support the stepwise maturation of T-cell precursors that colonize the thymus from the blood.In this study, we have investigated the mechanisms that regulate thymus growth and the availability of intrathymic epithelial niches that support normal numbers of developing T-cell precursors.We find that the epithelial compartment in the early fetal thymus contains a high proportion of proliferating cells, a finding that links initial thymus growth with an increase in thymic epithelial numbers.This phase of epithelial proliferation in the early thymus, which correlates well with a rapid increase in total thymocyte numbers at these stages (Penit and Vasseur 44 and data not shown), is likely to serve to increase the availability of intrathymic stromal niches required to  support the large numbers of immature thymocytes generated by expansion of the first waves of thymus-colonizing T-cell precursors.In line with this possibility, we show that although thymic epithelial differentiation and organization into functional cortical and medullary microenvironments occur normally in the absence of thymus growth, the thymus remains small and does not contain sufficient intrathymic niches to support normal numbers of immature thymocytes.This finding has important implications for our understanding of the mechanisms regulating initial thymus development, and potentially thymus regeneration, as it suggests that the mechanisms regulating proliferation and differentiation of epithelial progenitors are distinct.
By analyzing cellular compartments of the early embryonic thymus, we identified a correlation between declining epithelial   cell proliferation and the presence of PDGFR␣ ϩ mesenchyme cells.This suggests that the latter play an important role in providing proliferative signals to the thymic epithelium, a possibility supported by our finding that removal of PDGFR␣ ϩ mesenchyme prevented normal thymus growth in vivo.In addition, the presence in hypoplastic thymus grafts of adult host kidney capsulederived mesenchyme, shown directly using transplants of eYFP fetal donor thymus grafts, suggests that fetal thymic PDGFR␣ ϩ mesenchyme are specialized in their ability to regulate thymus growth.While the nature of this specialization is not fully understood, we have previously shown using an in vitro approach that at E14 of gestation, fetal thymic mesenchyme produces growth factors such as FGF7 and FGF10 that can induce proliferation of FGFR2iiib-expressing thymic epithelial cells, 38 a finding that correlates with reduced thymus size in FGFR2iiib-deficient mice. 45owever, the question of whether FGFs play a role in the expansion of recently described bipotent epithelial progenitors that are present in the E12 thymus lobes used in this study has not been addressed.Indeed, semiquantitative PCR analysis shows that E12 PDGFR␣ ϩ thymic mesenchyme lacks detectable FGF7 and FGF10 expression, while kidney capsule mesenchyme expresses readily detectable levels of FGF7 and FGF10, which argues against the notion that E12 thymic epithelial grafts fail to growth because of a lack of local FGFs.Taken together with our earlier study, 38 these observations suggest that while FGFs regulate proliferation of epithelial cells at later stages of development, a distinct mechanism regulates proliferation of bipotent epithelial progenitors in the E12 thymus.Indeed, in further analysis, we found that IGF1 and IGF2 mRNA detected in E12 PDGFR␣ ϩ fetal thymic mesenchyme, but not in adult kidney capsule mesenchyme, findings that correlate with the expression of IGF1-receptor mRNA by E12 thymic epithelium.Collectively, these observations suggest that IGF-IGF receptor interactions may play a role in the growth of the E12 thymus.Such a hypothesis is supported by the demonstration that IGFs play a role in the proliferation of epithelial cells in other tissues, such as the mammary gland, 42,43 as well as in increased thymus size reported in transgenic mice overexpressing IGF2. 468][49] Collectively, these findings support the notion that neural crest-derived mesenchyme represents a transient thymic cell type whose presence in the thymus is temporally regulated and that determines the initial phase of embryonic thymus growth.Although a small population of PDGFR␣ ϩ mesenchyme is still present in the neonatal and adult thymus (Figure 1; W.E.J., unpublished observations, November 2005), it remains to be established whether these cells play any role in regulating epithelial cell proliferation in the postnatal period.In this context, strategies to stimulate re-expansion or functional reactivation of this population could promote thymus recovery following ablative therapy or age/disease-related involution.Alternatively, intrathymic transplantation of PDGFR␣ ϩ mesenchyme and/or identification of the products mediating the effects of these cells on epithelial proliferation may also provide strategies to restore thymic growth and thereby T-cell output.For personal use only.on August 31, 2017.by guest www.bloodjournal.orgFrom

Figure 1 .
Figure 1.Thymic epithelial cells undergo a temporally regulated phase of cellular proliferation.Freshly dissected thymus lobes were disaggregated and analyzed by flow cytometry to detect expression of cytokeratin and the proliferation marker Ki67.(A) A typical example of an E14 thymus preparation, with double-positive cytokeratin ϩ Ki67 ϩ cells representing proliferating thymic epithelial cells.Figures in quadrants represent the percentage of the analyzed population.(B) The percentages of cytokeratin ϩ Ki67 ϩ cells present in the thymi of the indicated ages, together with the percentage of PDGFR␣ ϩ mesenchyme within the nonhemopoeitic nonepithelial (CD45 Ϫ EpCAM1 Ϫ ) thymic fraction.Results are averaged from at least 3 independent experiments, and are presented with standard deviations (error bars).

Figure 2 .
Figure 2. The E12 thymus anlagen has 3 distinct cellular compartments.E12 thymus lobes were disaggregated and analyzed by flow cytometry for expression of the panhemopoietic marker CD45 (A), and the panepithelial marker cytokeratin together with the mesenchyme marker PDGFR␣ (B).Note that most cells at this stage are CD45 Ϫ stromal cells, consisting of 2 dominant cytokeratin ϩ PDGFR␣ Ϫ epithelial and cytokeratin Ϫ PDGFR␣ ϩ mesenchymal subsets.Data shown are representative of 4 separate experiments.Figures in quadrants are representative of the percentage of the analyzed population.

Figure 3 .
Figure 3. Preparation of mesenchyme-free thymic epithelial rudiments.To separate the mesenchymal and epithelial components of the E12 thymus, lobes were incubated briefly in collagenase and then drawn up into mouth-controlled glass pipette (A), which shears surrounding mesenchyme, resulting in a smooth epithelial core (B) (arrowhead).*An unmanipulated E12 thymus lobe with surrounding mesenchyme still attached is shown for comparison (B).Compared with cell suspensions from whole E12 thymus lobes (C), epithelial rudiments prepared in this way are devoid of cells expressing the mesenchyme marker PDGFR␣ (D).Data shown are typical of 3 separate experiments.

Figure 4 .
Figure 4. Thymic epithelial progenitors generate functional and organized microenvironments in the absence of PDGFR␣-expressing fetal mesenchyme.Mesenchyme-free E12 thymus rudiments, placed under the kidney capsule for 3 weeks, were analyzed by immunohistochemistry for expression of the cortical epithelial marker cytokeratin 8 and the medullary epithelial marker cytokeratin 5 (A).Note that distinct cytokeratin 5 Ϫ 8 ϩ cortical and K5 ϩ 8 Ϫ medullary areas are present and are separated by a cortico-medullary junction (dotted line).Thymocytes harvested from whole (B,D) or mesenchyme-stripped (C,E) E12 thymus grafts were analyzed by flow cytometry for expression of CD4 and CD8, and the ␣␤ T-cell receptor.Results are representative of least 3 independent experiments.Figures in quadrants represent the percentage of the analyzed population.

Figure 5 .
Figure 5. PDGFR␣-expressing fetal mesenchyme regulates thymus growth and the availability of intrathymic niches.(A) Kidneys excised from mice that 3 weeks earlier received either whole unmanipulated (arrow) or mesenchyme-stripped (arrowhead) E12 thymus grafts under the renal capsule.Note the increased growth achieved in unmanipulated versus stripped thymus grafts (B).Importantly, grafts formed from epithelial cores reassociated with mesenchyme prior to transplantation were found to grow and contain similar thymocyte numbers in a manner comparable with unmanipulated thymus lobes.Grafts of unmanipulated of mesenchyme-stripped thymus lobes were analyzed for either thymocyte cellularity (C) or by immunohistochemistry using ERTR7 to identify host-derived mesenchyme that had invaginated the graft (D-E).Data shown are typical of 3 separate experiments.Results are averaged from at least 3 independent experiments, and are presented with standard deviations (error bars).

Figure 6 .
Figure 6.E12 thymus grafts contain host-derived mesenchyme.To determine the origin of mesenchymal cells present in thymus grafts, E12 thymus lobes from eYFP transgenic mice were transplanted into non-eYFP WT adult hosts (A).Grafts were harvested after 3 weeks, and mesenchyme cells were identified on the basis of an EpCAM1 Ϫ CD45 Ϫ phenotype to exclude all hemopoietic and epithelial cells, together with the markers Ly51 and MTS15.(B-C) Host-derived eYFP Ϫ MTS15 ϩ and eYFP Ϫ Ly51 ϩ mesenchyme cells are readily detectable in the thymus grafts.Data shown are representative of at least 3 separate experiments.

Figure 7 .
Figure 7. PDGFR␣ ؉ E12 thymic mesenchyme but not kidney capsule mesenchyme expresses insulinlike growth factors, which correlates with IGF1R expression by thymic epithelium.Semiquantitative RT-PCR was used to compare expression of FGF7 (A), FGF10 (B), IGF1 (C), and IGF2 (D) in E12 PDGFR␣ ϩ thymic (f) and adult kidney capsule mesenchyme (Ⅺ).In addition, IGF1-receptor expression (E) was analyzed in thymic epithelium (f) and PDGFR␣ ϩ mesenchyme (f) from E12 thymus lobes.Note that expression of IGF1 and IGF2 mRNA by fetal thymic mesenchyme correlates with IGF1R expression by thymic epithelium.Data shown are representative of 3 separate experiments and are expressed as mean Ϯ SEM.