Three Chemokine Receptors Cooperatively Regulate Homing of Hematopoietic Progenitors to the Embryonic Mouse Thymus

Three chemokine receptors cooperatively regulate homing of hematopoietic progenitors to the embryonic mouse thymus

Lesly Calderón and Thomas Boehm1

Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, D-79108 Freiburg, Germany

Edited* by Max D. Cooper, Emory University, Atlanta, GA, and approved March 25, 2011 (received for review November 2, 2010) The thymus lacks self-renewing hematopoietic cells, and thymopoi- These results were interpreted as indicating that Ccr9/Ccl25 and esis fails rapidly when the migration of progenitor cells to the Ccr7/Ccl21 are essential only for the prevascular stage of thymus thymus ceases. Hence, the process of thymus homing is an essen- colonization. Interestingly, two recent reports demonstrate that tial step for T-cell development and cellular immunity. Despite de- adult mice lacking both Ccr7 and Ccr9 display severe reductions cades of research, the molecular details of thymus homing have not in the number of early thymic progenitors and suggest that been elucidated fully. Here, we show that chemotaxis is the key compensatory expansion of intrathymic populations could ex- mechanism regulating thymus homing in the mouse embryo. We plain, at least in part, normal thymic cellularity (13, 14). Although determined the number of early thymic progenitors in the thymic these studies ascribe important roles to Ccr9 and Ccr7 in thymus rudimentsofmicedeficient for one, two, or three of the chemokine colonization, these receptors do not appear to be essential, sug- receptor genes, chemokine (C-C motif) receptor 9 (Ccr9), chemokine gesting that other molecules might be involved, for instance (C-C motif) receptor 7 (Ccr7), and chemokine (C-X-C motif) receptor 4 Cxcl12 and its receptor Cxcr4. In initial analyses of Cxcr4- and (Cxcr4). In the absence of all three chemokine receptors, thymus Cxcl12-deficient mice, no defect in T-cell development was ob- homing was reduced about 100-fold both before and after vascu- served (15–17), although it was found subsequently that thymo- larization of the thymic rudiment. In the absence of only two of cyte numbers were reduced at later stages of embryonic devel- these three chemokine receptor genes, thymus homing was much opment (18, 19), and this reduction recently was attributed to less affected (only two- to 10-fold), indicating that the chemotactic the contribution of the Cxcr4/Cxcl12 signaling pathway to effi- regulation of thymus homing is remarkably robust. Our results re- cient β selection (19, 20). In medaka (Oryzias latipes) and zebra- veal the redundant roles of Ccr9, Ccr7, and Cxcr4 for thymic homing fish (Danio rerio) embryos, simultaneous interference with ccl25a and provide a framework to examine the regulation of progenitor and cxcl12 specifically and completely blocked thymopoiesis, homing in the postnatal thymus. suggesting that cxcr4/cxcl12 and ccr9/ccl25 cooperate in guiding progenitors to settle in the thymus of fish embryos (21). -cell development occurs in the thymus. Beginning at midg- In this study, we therefore examined the relative contributions Testation (1), lymphocyte progenitors migrate to the thymus of Cxcr4, Ccr9, and Ccr7 chemokine receptors in the homing of IMMUNOLOGY anlage (2, 3), and there they develop into mature T cells that T-cell progenitors to the mouse embryonic thymic rudiment. emigrate to the periphery. The colonization of the thymic rudi- ment is the essential starting point of thymopoiesis; without this Results colonization, intrathymic T-cell development does not occur. Combined Absence of Ccr9 and Cxcr4 Chemokine Receptor Genes More than 30 y ago, chemotactic mechanisms were proposed to Does Not Abolish Thymopoiesis. In lower vertebrates, the co- underlie the thymus homing process (4, 5). However, so far, no operative activities of Ccr9/Ccl25 and Cxcr4/Cxcl12 chemokine single factor has been shown to regulate this process, leading to receptor/chemokine pairs regulate thymus homing (21). To the suggestion that the combinatorial activity of many factors is examinewhetherthesameistrueforthymushomingin required (6). The nature of these factors has not yet been defined. mammals, we generated mice deficient for both Ccr9 and Cxcr4. From the earliest stages of thymus development [embryonic Because it is not possible to perform live imaging of intact day (E) 11.5–12.5] onwards, chemokines such as chemokine (C- mouse embryos, we used the number of thymocytes in the C motif) ligand 21 (Ccl21), chemokine (C-C motif) ligand 25 thymic rudiments at E14.5 and 17.5 as a surrogate parameter of (Ccl25), and chemokine (C-X-C motif) ligand 12 (Cxcl12) are thymus homing. E17.5 was the latest time point that could be expressed in the thymic anlage (7–10). Cxcl12 also is expressed in examined reliably because of embryonic lethality of Cxcr4-de- the tissue surrounding the thymus, the perithymic mesenchyme ficient mice (16, 17, 22). Unexpectedly, thymopoiesis was not (7, 10); in addition, Ccl21 is expressed in the primordium of the completely abolished in Ccr9;Cxcr4 double-deficient embryos. parathyroid (7–10). At E12.5, the receptors for Ccl21, Ccl25, and AsshowninFig.1,Ccr9 deficiency alone led to only a slight Cxcl12 chemokines (Ccr7, Ccr9, and Cxcr4, respectively) are reduction in the number of thymocytes at E14.5 and E17.5, expressed in CD45+ cells isolated from the perithymic mesen- as described previously (8, 11); the effect of Cxcr4 deficiency chyme (10). on thymocyte numbers was more pronounced, particularly at Several studies support the notion that chemokines and their E17.5. Although thymocyte numbers were reduced more sub- receptors play a role in the thymus homing process. Ccr9-deficient stantially in Ccr9;Cxcr4 double-deficient embryos (by about embryos display a reduced thymocyte number at early, but not 3.4-fold at E14.5 and 9.3-fold at E17.5) than in embryos de- later, stages of thymus development (8, 11, 12). Paucity of lymph node T cells (plt)/plt embryos, which lack functional chemokine (C-C motif) ligand 19 (Ccl19) and Ccl21 genes, and Ccr7-deficient Author contributions: L.C. and T.B. designed research; L.C. performed research; L.C. and embryos exhibit an early impairment of thymus colonization but T.B. analyzed data; and L.C. and T.B. wrote the paper. have attained normal thymic cellularity by E14.5 (9). Compound The authors declare no conflict of interest. Ccr9 and Ccr7 deficiency leads to an even stronger defect; at *This Direct Submission article had a prearranged editor. −/− −/− E11.5, thymic primordia of Ccr9 ;Ccr7 embryos are poorly 1To whom correspondence should be addressed. E-mail: [email protected]. colonized by hematopoietic cells. Nonetheless, these effects are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transient, because thymocyte numbers are normal at birth (8). 1073/pnas.1016428108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1016428108 PNAS | May 3, 2011 | vol. 108 | no. 18 | 7517–7522 Downloaded by guest on October 2, 2021 Total thymocytes all thymocytes express the cell-surface marker CD45 (24, 25); 7 E14.5 thus, these cells most likely include the thymic immigrants and E17.5 ) their immediate progeny. The epithelium of organ primordia

10 6 is positive for cytokeratin 8; therefore, sagittal sections were immunostained for cytokeratin 8 and CD45 to reveal the precise positions of hematopoietic cells (Fig. 2). In control embryos 5 +/− +/− +/− (Ccr9 ;Ccr4 ;Ccr7 ), CD45+ cells were located inside and around the thymic primordium but also around the parathyroid −/− +/− 4 anlage (Fig. 2A). In Ccr9-deficient embryos (Ccr9 ;Cxcr4 ;

Cell Number (log +/− Ccr7 ) reduced numbers of cells were found within the thymus 3 but not around the parathyroid (Fig. 2B); in contrast, the dis- +/− −/− n 15 9 21 11 7 2 7 9 tribution of cells in Cxcr4-deficient embryos (Ccr9 ;Cxcr4 ; +/− Ccr9 +/- -/- +/- -/- Ccr7 ) appeared similar to that in controls (Fig. 2C). This Cxcr4 +/- +/- -/- -/- distribution is in keeping with the results shown in Fig. 1 and with previous observations (18). In embryos deficient for both Ccr9 Fig. 1. Combined lack of Ccr9 and Cxcr4 chemokine receptors does not −/− −/− +/− and Cxcr4 (Ccr9 ;Cxcr4 ;Ccr7 ), CD45+ cells were found abolish intrathymic T-cell development. The number of thymocytes is shown D fi for embryos of the indicated genotypes at E14.5 and E17.5. The number of mainly around the parathyroid anlage (Fig. 2 ). These ndings analyzed embryos is indicated (n). are compatible with the notion that the high levels of Ccl21 expression in the parathyroid contribute to the attraction of progenitors to the perithymic area (7, 9). Accordingly, the +/− +/− −/− ficient for either chemokine receptor gene alone, significant distribution of CD45+ cells in Ccr9 ;Cxcr4 ;Ccr7 embryos numbers of thymocytes were still detectable at both stages ex- is characterized by the conspicuous absence of hematopoietic amined (Fig. 1). These results differ from those observed in fish cells near the parathyroid and reduced numbers within and (21) and indicate that the regulation of thymus homing in mice around the thymus (Fig. 2E), a reciprocal distribution compared −/− −/− +/− −/− is more complex than in lower vertebrates. with Ccr9 ;Cxcr4 ;Ccr7 embryos (Fig. 2D). In Ccr9 ; +/− −/− +/− −/− −/− Cxcr4 ;Ccr7 and Ccr9 ;Ccr4 ;Ccr7 embryos, the thy- Chemotaxis Regulates the Distribution of Hematopoietic Precursors mus still contained CD45+ cells, albeit in reduced numbers (Fig. in the Proximity of the Common Parathyroid/Thymus Anlage. Pre- 2 F and G), showing that Ccr9 and Cxcr4 on their own are suf- vious work in mice has suggested a role for the Ccr7 chemokine ficient to sustain the homing process. Notably, triple-deficient −/− −/− −/− receptor in thymus homing (8, 9, 13, 14). Therefore, we pro- embryos (Ccr9 ;Cxcr4 ;Ccr7 ) lacked CD45+ cells in and ceeded to examine the additional role of Ccr7 in the context of around the primordia of the thymus and the parathyroid. Col- single and combined deficiencies of Ccr9 and Cxcr4. To this end, lectively, these results suggest that chemotaxis regulates the dis- + we examined the distribution of CD45 hematopoietic cells tribution of progenitor cells in and around the thymus. in and around the thymic anlage by immunohistochemistry. At E12.5, the epithelial anlagen of the thymus and parathyroid have Thymopoiesis in Ccr9;Cxcr4;Ccr7 Triple-Deficient Mice Is Severely budded off the pharynx but have not yet fully separated into Impaired. These results suggest that all three chemokine recep- individual organ primordia (23). Hence, this time point affords tors contribute to thymus homing, albeit in different ways. To a comprehensive analysis of the distribution of hematopoietic assess the effect of triple deficiency on thymopoiesis quantitatively, progenitors in and around these organs. Our breeding scheme we determined the number and phenotype of thymocytes in the generated the expected 27 genotypes in nearly normal Mende- thymic rudiments of E14.5 mice by flow cytometry. In heterozy- lian ratios up to E17.5. For the sake of simplicity, only the results gous control mice, about 20,000 cells were recovered, whereas in for heterozygous and nullizygous genotypes (n = 8) are reported triple-deficient mice, only about 400 cells were obtained (about here; hence, mice heterozygous at all three loci provide the 2% of control values; Fig. 3A). At this time point, thymopoiesis baseline reference in the experiments described below. At E12.5, already has begun, and the differentiation of thymocytes has

Ccr9+/- Cxcr4+/- Ccr7+/- Ccr9-/- Cxcr4+/- Ccr7+/- Ccr9+/- Cxcr4-/- Ccr7+/- Ccr9-/- Cxcr4-/- Ccr7+/- A BCD

Ccr9+/- Cxcr4+/- Ccr7-/- Ccr9-/- Cxcr4+/- Ccr7-/- Ccr9+/- Cxcr4-/- Ccr7-/- Ccr9-/- Cxcr4-/- Ccr7-/- E F GH

Fig. 2. Spatial distribution of CD45+ cells in the pharyngeal region of E12.5 embryos. Cryosections were stained for cytokeratin 8 (green fluorescence) and CD45 (red fluorescence). The boundaries between the anlagen of the parathyroid (pointing to the upper left corner) and the thymus (lower right) are in- dicated by the dashed lines. The genotypes of embryos are indicated above A–H. The panels are representative of two to four thymic lobes. (Scale bar, 50 μm.)

7518 | www.pnas.org/cgi/doi/10.1073/pnas.1016428108 Calderón and Boehm Downloaded by guest on October 2, 2021 A 5 double deficiency was observed with Ccr7 and Ccr9 mutants Total thymocytes (about 5% of control values). From these results, we infer that )

10 Ccr9 is the most important factor in regulating thymus homing 4 at this stage. The results for ETPs were qualitatively similar, al- though the effects tended to be more pronounced (Fig. 3C). When immature thymocytes were defined by a different cell surface − marker combination [Lin CD117+/Paired immunoglobulin re- 3 ceptor (PIR+)] (27), similar findings were made (Fig. S1). Col- Cell Number (log lectively, our results indicate that at E14.5, the thymic rudiment in triple-deficient mice lacks T-cell progenitors. 2 n 21 12 4513 10 7 7 fi Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- Triple-De cient Mice Possess the Same Number of T-Cell Progenitors Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- in the Fetal Liver. Instead of being the result of impaired homing, Ccr7 +/- +/- +/- +/- -/- -/- -/- -/- the lack of T-cell progenitors in the thymic anlage of mutant mice might have been caused by a failure in their production in the fetal liver. To examine this possibility, we determined the 13.4 13.8 B numbers of various progenitor populations in this tissue. Cxcr4 deficiency always causes a 30% reduction in the numbers of total 92.5 − fetal liver cells (Fig. 4A). The Lin CD117+/ stem cell antigen 1 Lin + CD117 (Sca-1 ) population, enriched in multipotent progenitors (28), was affected only marginally, as were the mostly lymphoid- − primed Lin CD117+ Sca-1+/FMS-related tyrosine kinase 3 FSC-A CD25 (Flt3)hi multipotent progenitors (29) (Fig. S2 A–C). The − Lin CD117+CD127+ population in fetal liver (Fig. 4B) is en- Lin-CD117+CD25- riched in progenitors with B and T/natural killer (NK) cell po- C − 4000 30 tential, respectively (30), which can be assigned to PIR and + − + + − 20 PIR subpopulations: the Lin CD117 CD127 PIR subset 3000 − + + + 10 contains B-cell progenitors, and the Lin CD117 CD127 PIR 2000 0 subset contains exclusively T/NK/dentritic cell progenitors (27). In keeping with these and previous findings (15–17, 31, 32), 1000 − + + − 800 Lin CD117 CD127 PIR cells were decreased drastically in all Cxcr4-deficient embryos, but the absence of the other chemokine Cell Number 400 receptor genes had no significant influence (Fig. 4C). By con- − trast, the effect of Cxcr4 deficiency on Lin CD117+CD127+ 0 PIR+ cells (henceforth designated “PIR+ cells”) was much less n 20 11 3 5 11 10 7 7 D Ccr9 fi IMMUNOLOGY Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- pronounced (Fig. 4 ). Although de ciency alone did not + Ccr7 Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- appear to have any effect on PIR cells, lack of increased + Ccr7 +/- +/- +/- +/- -/- -/- -/- -/- their number ∼1.5-fold; the number of PIR cells was normal- ized in embryos double-deficient for Ccr7 and Cxcr4, irrespective Fig. 3. T-cell progenitors in the thymus of E14.5 embryos deficient for of the Ccr9 genotype (Fig. 4D). To examine the capacity of chemokine receptors. (A) Total number of thymocytes in embryos of the mutant PIR+ cells to generate T cells in vitro, they were cultured − indicated genotypes. (B) Representative flow cytometric profile of a Ccr9+/ ; on OP9-DL1 stromal cells (33). Although deficiency of Cxcr4 +/− +/− fi Ccr7 ;Cxcr4 embryo to illustrate the identi cation of ETPs. Numbers caused a reduction in cell numbers after 8 d of in vitro growth, this +/− −/− represent percentages of total thymocytes. (C) Number of ETPs in embryos inhibition was comparable for PIR+ cells of Ccr9 ;Cxcr4 ; of the indicated genotypes. Note that the scale is expanded in the lower part Ccr7+/− Ccr9−/− Cxcr4−/− Ccr7−/− of the y axis; the inset is a magnification to show the absolute number of and ; ; embryos. Importantly, how- ETPs for the three color-coded genotypes. Average values are indicated; ever, no qualitative defect in T lineage differentiation, as mea- each data point represents one embryo, and the total number of embryos sured by the number of DN2/3 cells, was detectable (Fig. 4E). The − + + per genotype is indicated (n). same is true when Lin CD117 Sca-1 cells were subjected to in vitro differentiation (Fig. S2D). Collectively, these data suggest −/− that T-cell progenitors are generated in the fetal liver of Ccr9 ; + + − −/− −/− progressed from the DN1 (CD45 CD44 CD25 ) to the DN4 Cxcr4 ;Ccr7 embryos in nearly normal numbers and that they + − − stage (CD45 CD44 CD25 ). The DN1 population contains early are capable of differentiating normally into T cells in vitro. thymic progenitors (ETPs), defined by the presence of c-kit (CD117) and the absence of lineage markers, including CD25 Thymus Homing in Triple-Deficient Mice Does Not Resume After (26). When we determined the number of ETPs [lineage marker- Vascularization. These experiments addressed thymopoiesis at − − negative (Lin )CD117+CD25 ](Fig.3B), about 2,400 ± 800 cells the prevascularized stage of thymus development. It has been were found in the heterozygous controls (Fig. 3C). By contrast, argued, however, that vascularization affects the homing pro- fewer than 10 such cells could be found in the triple-deficient cess (8). Hence, it was important to determine whether the thymic rudiments (Fig. 3C). The thymocyte numbers in single- and observed effects on thymopoiesis persist beyond E14.5. To this double-deficient mice again suggest that the relative contributions end, we examined the state of thymopoiesis at E17.5, well be- of the three chemokine receptors differ (Fig. 3A). Loss of Cxcr4 yond the onset of vascularization (Fig. 5 A–E). The total function reduced thymocyte numbers by about 30%; loss of Ccr7 number of thymocytes in triple-deficient mice (about 3,000 function had a slightly more pronounced effect (a reduction of cells) was lower by more than two orders of magnitude than in about 40%). The strongest effect was seen with Ccr9 deficiency, controls (about 1,000,000 cells) and by at least one order of which reduced thymocyte numbers by 85%; the same level of re- magnitude than in other mutant embryos (Fig. 5F). The dis- duction was seen in combined deficiency of Ccr7 and Cxcr4. tribution of CD45+ cells found within the epithelium (Fig. 5E) Combined deficiency of Ccr9 and Cxcr4 reduced thymocyte appears to be essentially random with respect to cortical and numbers to about 10% of control values. The strongest effect of medullary areas (Fig. S3). The number of ETPs in the thymic

Calderón and Boehm PNAS | May 3, 2011 | vol. 108 | no. 18 | 7519 Downloaded by guest on October 2, 2021 3 A 3 D - + + + Total cells Lin CD117 CD127 PIR ) ) 4 7 2 2

1 1 Cell Number (10 Cell Number (10

0 0 n 28 10 7 4 16 15 6 9 n 28 10 7 4 16 15 6 9 Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- Ccr7 +/- +/- +/- +/- -/- -/- -/- -/- Ccr7 +/- +/- +/- +/- -/- -/- -/- -/-

6 B 25.4 E Total cells

) 5 DN2+DN3 0.44 0.05 4 4 CD127 CD117 3

2

Lin PIR Cell Number (10 1 Lin-CD117+CD127+PIR- C 2.0 0 n 63436625 ) 5 1.5 Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- Ccr7 +/- +/- +/- +/- -/- -/- -/- -/- 1.0

0.5 Cell Number (10

0.0 n 28 10 7 4 16 15 6 9 Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- Ccr7 +/- +/- +/- +/- -/- -/- -/- -/-

Fig. 4. Early progenitor cells in the fetal liver of E14.5 embryos. (A) Total number of cells for embryos of the indicated genotypes. (B) Representative flow − − − − − − cytometric profile of a Ccr9+/ ;Ccr7+/ ;Cxcr4+/ embryo to illustrate the identification of Lin CD117+CD127+PIR and Lin CD117+CD127+PIR+ populations. − − Numbers refer to percentages of total cells. (C) Number of Lin CD117+CD127+PIR cells in the fetal liver of embryos with the indicated genotypes. (D) Number of Lin−CD117+CD127+PIR+ cells. (E) Total number of cells and number of DN2 + DN3 cells obtained after in vitro culture of 100 Lin−CD117+CD127+PIR+ cells for 8 d on OP9-DL1 stroma. The total number of embryos analyzed per genotype is indicated (n).

lobes mirrored this situation; in triple-deficient embryos, only thymic lobes by wild-type host cells was not impaired (Fig. S5D). about 50 ETPs per thymus were found, whereas about 5,000 Collectively, these results demonstrate that, although the mutant cells of this phenotype were observed in controls (Fig. 5 G and cells are severely impaired in their ability to colonize the thymus, H). The same is true when early progenitors are defined as they nonetheless are capable of progressing through all stages of − Lin CD117+PIR+ cells (Fig. S4). Hence, even after vascular- T-cell development. ization, the number of ETPs remains at extremely low levels in triple-deficient thymi, suggesting that the combined functions Discussion of three chemokine receptors account for >99% of homing Here we show that chemotaxis is the key mechanism regulating activity during the entire embryonic period. thymus homing in mouse embryos before and after vasculariza- To exclude the possibility that the absence of all three che- tion of the thymus. This chemotactic regulation is remarkably mokine receptors affected the general physiology of thymocytes robust because of the redundant activities of the Ccr9, Ccr7, and in a way that interfered with their normal intrathymic development, Cxcr4 chemokine receptors. E17.5 embryos were subjected to further analyses. The presence of Our results indicate that the presumptive T-cell progenitor − more mature DN thymocyte populations (Lin CD117+CD25+ populations, each identified by a characteristic combination of − and Lin CD117-/loCD25+) in mutant thymi indicated that the cell surface markers, are present in normal numbers in the fetal triple-deficient cells can develop further (Fig. S5A). When thymic liver of triple-deficient embryos. Because they neither accumu- lobes were removed from the embryos and allowed to develop late in nor disappear from the fetal liver, it seems unlikely that further in fetal thymus organ cultures for 8 d, CD4/CD8 double- they are functionally incapacitated. Indeed, when assayed in positive thymocytes developed (Fig. S5B). The same result was vitro, these cells are capable of differentiating into T cells. Al- obtained when the thymic lobes were transplanted under the though we have not been able to isolate T-cell progenitors di- kidney capsule of syngeneic wild-type mice and analyzed 12 rectly from fetal blood, it appears unlikely that their ability to d later (Fig. S5C). Moreover, the colonization of triple-deficient enter the circulation and to survive in this environment is af-

7520 | www.pnas.org/cgi/doi/10.1073/pnas.1016428108 Calderón and Boehm Downloaded by guest on October 2, 2021 Ccr9+/- Cxcr4+/- Ccr7+/- Ccr9-/- Cxcr4-/- Ccr7+/- Ccr9-/- Cxcr4+/- Ccr7-/- Ccr9+/- Cxcr4-/- Ccr7-/- Ccr9-/- Cxcr4-/- Ccr7-/- A B C D E

F 7 GH12,000 Total thymocytes Lin-CD117+CD25-

) 9,000

10 6 6,000

Lin 3,000 5 1,000 39.1 4 600 FSC-A 200 Cell Number Cell Number (log 3 0.63 1.0 100 50 2 0 n 191037181575 n 18 9 3 7 16 15 6 5

Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- CD117 Ccr9 +/- -/- +/- -/- +/- -/- +/- -/- Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- Cxcr4 +/- +/- -/- -/- +/- +/- -/- -/- Ccr7 +/- +/- +/- +/- -/- -/- -/- -/- Ccr7 +/- +/- +/- +/- -/- -/- -/- -/- CD25

Fig. 5. T-cell progenitors in the thymus of E17.5 embryos deficient for chemokine receptors. (A–E) Distribution of CD45+ cells in the thymic rudiment of E17.5 embryos. The genotype is indicated at the top of each panel. Cryosections were stained for cytokeratin 8 (epithelial marker, green fluorescence), CD45 (marker of hematopoietic cells, red fluorescence), and CD31 (endothelial marker, blue fluorescence). The panels are representative of two to six thymic lobes. − − (Scale bar, 100 μm.) (F) Number of total thymocytes in embryos of the indicated genotypes. (G) Representative flow cytometric profile of a Ccr9+/ ;Ccr7+/ ; − Cxcr4+/ embryo to illustrate the identification ETPs. Numbers refer to percentages of total thymocytes. (H) Number of ETPs in embryos of the indicated genotypes. Note that the scale is expanded in the lower part of the y axis. The total number of embryos per genotype is indicated (n).

fected by the lack of chemokine receptors, because neither Ccr7 arrive in the pharyngeal region via the blood stream, where they

nor Ccr9 has been implicated in these processes (8). Cxcr4 is respond to chemokines emanating from the rudiments of the IMMUNOLOGY known to regulate, among other processes, the adhesion of he- thymus and the parathyroid. Although they are heterogeneous matopoietic cells to the stromal microenvironment (32). Hence, with respect to chemokine receptor expression (10), they none- progenitors arriving in the thymus might be nonspecifically lost theless represent a developmentally homogeneous population because of their diminished retention. However, we note that (34). Because Ccl25 and Cxcl12 (both of thymus origin) are con- such cells are found in large numbers in the thymus of Cxcr4;Ccr7 siderably more effective in attracting progenitor cells than is Ccl21 and Cxcr4;Ccr9 double-deficient embryos. Therefore, we believe (predominantly of parathyroid origin) (7–10), progenitor cells that the lack of thymocytes in triple-deficient embryos is a direct collect around and in the thymic rudiment rather than near the result of impaired responses to chemokine gradients emanating parathyroid. Hence, progenitor accumulation in the vicinity of the from the thymic microenvironment. Indeed, intrathymic devel- thymus of early embryos is most effective when all three chemo- opment of the few progenitors arriving in the thymus of triple- kine receptors are functionally competent. Once thymus and deficient embryos into mature T cells is not impaired. parathyroid have become two separate organs and have located to Previous studies on thymus homing have focused on the roles different regions of the embryo, the importance of parathyroid- of Ccr9 and Ccr7, whereas the potential function of Cxcr4 in this derived Ccl21 for thymus homing diminishes; however, this che- process has been largely ignored. We were prompted to analyze mokine continues to be expressed by the thymic epithelium and the contribution of Cxcr4 to thymus homing in mice following contributes to thymus homing. As a result of these redundancies, our demonstration that thymus homing in fish could be abolished homing to the thymus is remarkably robust. Our results do not by simultaneously interfering with ligands for Ccr9 and Cxcr4 exclude the possibility that other molecules apart from chemo- (21). Our present analysis supports the notion that chemotactic kines and their receptors are involved in the homing process. regulation of thymus homing is a conserved feature of vertebrate However, their direct involvement during the embryonic phase is thymus function and that this process has evolved to become likely to be marginal or indirect and then possibly dependent on more redundant and robust in mammals. Several factors con- intracellular signals emanating from chemokine receptors (35). tribute to this enhanced redundancy. First, in contrast to the situation in fish, Cxcl12 is expressed not only by the capsular Materials and Methods mesenchyme but also by the thymic epithelium. Second, thymus Animals. Mouse strains deficient for Ccr9, Ccr7, and Cxcr4 have been de- homing is helped, at least initially, by the close proximity of the scribed (12, 17, 36). Ccr7-deficient mice with the C57BL/6 genetic background thymus to the parathyroid, which expresses copious amounts of were obtained from Jackson Laboratories, and Ccr7-deficient mice with the BALB/c background were obtained from M. Lipp (Max Delbrück Center for the chemokine Ccl21, the ligand for the Ccr7 chemokine receptor. fi fi Ccr9−/− Cxcr4−/− Molecular Medicine, Berlin). Cxcr4-de cient mice were from Jackson Labo- Ccl21 appears to attract, albeit inef ciently, ; - fi fi ratories. Mice were kept and bred under speci c pathogen-free conditions de cient progenitors to the vicinity of the thymic rudiment, where at the animal facility of the Max Planck Institute of Immunobiology and some might come under the influence of “thymic” Ccl21. Epigenetics in Freiburg, Germany. Genotyping was performed as described Our data suggest the following sequence of events of thymus in SI Materials and Methods. Embryos were obtained from timed pregnan- homing during embryogenesis. At early stages, prothymocytes cies; the day of the vaginal plug was counted as day 0.5 of pregnancy.

Calderón and Boehm PNAS | May 3, 2011 | vol. 108 | no. 18 | 7521 Downloaded by guest on October 2, 2021 Immunohistochemistry. E12.5 and E17.5 embryos were embedded in optimal J. C. Zuniga-Pflücker, University of Toronto, Toronto) were plated at 2 × 103 cutting temperature (OCT) medium and snap frozen. E12.5 sagittal and E17.5 cells per well in a 96-well plate 48 h before the addition of progenitor cells. μ fi transversal sections (7- m thickness) were xed in acetone/methanol (3:1; vol/vol). Progenitors were cultured in the presence of recombinant murine IL-7, Flt3L, The following antibodies and reagents were used for staining: anti-cytokeratin and stem-cell factor (SCF) (5 ng/mL each; PeproTech) (37). Cells were ana- 8 [Troma-1; a gift from R. Kemler (Max Planck Institute of Immunobiology and lyzed by flow cytometry after 8 d in culture. Epigenetics, Freiburg, Germany)], allophycocyanin-conjugated anti-CD45 (30- F11; eBioscience), biotinylated anti-CD31 (MEC 13.3; BD Bioscience), Alexa Fluor Fetal Thymus Organ Culture and Thymus Transplantations. Fetal thymus organ 488 goat anti-rat IgG (Invitrogen), and Cy3-conjugated streptavidin (Jackson ImmunoResearch). Fluorochrome-labeled antibodies were detected using a Zeiss culture and thymus transplantations were carried out as described pre- AxioImager.Z1 microscope with ApoTome slider and Axiovision 4.8 software. viously (37, 38).

Flow Cytometric Analysis and Cell Sorting. Embryonic thymi were minced Statistical Analysis. Average values and SDs are shown. carefully with two thin forceps to prepare single-cell suspensions. Thymocyte numbers were determined using CountBright absolute counting beads ACKNOWLEDGMENTS. We thank Drs. Conrad Bleul and Martin Lipp for fl (Invitrogen). Analytical cytometry was performed using an LSRII, and cell mice, and J. C. Zuniga-P ücker for OP9-DL1 cells. We are grateful to J. B. sorting was done using a FACSAria (BD Biosciences). Swann for advice and critical reading of the manuscript. Financial support for these studies was provided by the Max Planck Society and the Deutsche Forschungsgemeinschaft. L. C. is a member of the International Max Planck − + + + OP9-DL1 Culture. E14.5 fetal liver progenitors (Lin CD117 CD127 PIR at 100 Research School for Molecular and Cellular Biology, a joint international PhD − + + cells per well or Lin CD117 Sca-1 at 200 cells per well) were sorted directly program of the Max Planck Institute of Immunobiology and Epigenetics and onto an OP9-DL1 layer in 96-well plates. OP9-DL1 cells (kindly provided by the University of Freiburg, Germany.

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