Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.


Introduction
The thymus medulla epithelium provides a unique environment for newly generated T cells to acquire self-tolerance before their emigration to the circulation.A variety of medullary thymic epithelial cells (mTECs) contribute to displaying self-antigens by employing multiple mechanisms.Promiscuous gene expression by a subpopulation of mTECs that express the nuclear protein Aire allows chromatin remodeling and genome-wide ectopic gene transcription (Derbinski et al., 2001;Anderson et al., 2002).Cellular mimesis in other mTEC subpopulations relies on a mosaic of tissue-specific transcription factor-dependent cells that individually resemble specialized cell types, including mucosal tuft cells and microfold cells (Farr et al., 2002;Michelson et al., 2022).These mTEC subpopulations form a self-antigen-displaying mTEC subset that provides various self-components, including extrathymic tissue-specific molecules, in the thymus medulla, so that newly generated T cells have an opportunity to establish central tolerance to genome-wide and systemic self-antigens prior to export from the thymus.
In addition to the self-antigen-displaying mTEC subset, there exists a medullary epithelial subset that produces the C-C chemokine CCL21, which attracts developing thymocytes and dendritic cells to the medullary region (Takahama et al., 2017).Positive selection-inducing TCR signals in newly generated cortical thymocytes elevate the expression of CCR7, a receptor for CCL21, and positively selected cortical thymocytes are therefore specifically attracted to migrate to the medullary region through the CCL21-CCR7-mediated chemotactic signals (Ueno et al., 2004;Takahama, 2006).Two molecular species CCL21Ser and CCL21Leu with one amino acid difference are encoded in the mouse genome (Nakano and Gunn, 2001;Chen et al., 2002), and CCL21Ser encoded by Ccl21a locus is predominantly expressed in the thymus medulla (Kozai et al., 2017).In mice lacking either Ccr7 or Ccl21a, positively selected mature thymocytes fail to accumulate in the medulla, and therefore T cells fail to establish self-tolerance (Kozai et al., 2017;Kurobe et al., 2006;Nitta et al., 2009).Additional CCR7-ligand CCL19 has no appreciable role in the cortex-to-medulla migration of developing thymocytes (Kozai et al., 2017;Link et al., 2007).Thus, CCL21-expressing thymocyte-attracting mTECs represent a functional mTEC subset that is essential for the establishment of self-tolerance in T cells.
CCL21-expressing mTECs are mostly included in the mTEC low subpopulation (Lkhagvasuren et al., 2013).However, how the CCL21-expressing mTEC subset is integrated with, and/or potentially diverted from, the developmental progression of the self-antigen-displaying mTEC subset remains unknown.A single-cell RNA-sequencing-based cluster analysis reported that Ccl21a transcripts are detectable within a heterogeneous 'intertypical' TEC subpopulation, which includes cells with gene expression profiles resembling immature mTEC progenitors (Baran-Gale et al., 2020).A more recent study has indicated that postnatally appearing mTEC-biased progenitors include cells that transcribe Ccl21a (Nusser et al., 2022).However, an independent single-cell RNA-sequencing-based study suggested that TECs expressing Ccl21a transcripts represent a terminal population derived from 'transit-amplifying' mTEC progenitors, by branching out of the self-antigen-displaying mTEC lineage (Wells et al., 2020).Without the direct evaluation of developmental potential, it remains controversial and unknown how CCL21-expressing mTECs are developmentally related to immature mTEC progenitors.It is even unclear how the Ccl21a-transcript-detectable TECs reported in these single-cell transcriptomic studies are related to functionally relevant CCL21-protein-producing medullary TECs.More specifically, it remains unestablished whether the functionally relevant CCL21-expressing mTEC subset retains a developmental potential to give rise to the self-antigen-displaying mTEC subset.
The present study addresses whether the CCL21-expressing mTEC subset carries a developmental capability to differentiate into the self-antigen-displaying mTEC subset.We show that CCL21-expressing mTECs arise early during embryonic development in mice.By Cre-loxP-mediated fate-mapping analysis, we demonstrate that a vast majority of self-antigen-displaying mTEC subset, including Aire-expressing mTECs and thymic tuft cells, originates from CCL21-expressing cells.The developmental potential of CCL21-expressing embryonic mTECs to give rise to Aire-expressing mTECs is confirmed in reaggregate thymus experiments.These results indicate that functionally relevant CCL21-expressing mTEC subset contains a developmental capability to differentiate into self-antigen-displaying mTEC subset and that developmental conversion from thymocyte-attracting subset to self-antigen-displaying subset serves to assemble functional diversity in thymus medulla epithelium.
Analysis of embryonic thymus showed that CCL21-expressing cells in the central region of the thymus were prominent by embryonic day 15 (E15) (Figure 2A) and detectable as early as E13 (Figure 2B).The localization of CCL21-expressing cells in the central region of embryonic thymus lobes and their expression of Krt14 and Foxn1 ascertained that CCL21-expressing embryonic thymus cells represented mTECs (Figure 2A-C), rather than neighboring CCL21 + parathyroid gland cells (Figure 2B), which contribute to the attraction of hematopoietic cells to the pre-vascularized embryonic thymus (Liu et al., 2006).CCL21 proteins were detectable in the medullary region proximal to Ccl21a tdTomato -expressing mTECs in the embryonic thymus by E15 (Figure 2D), indicating that Ccl21a tdTomato -expressing E15 embryonic mTECs represent functionally relevant CCL21-expressing mTECs.On the other hand, CCL21-expressing mTECs in the embryonic thymus were largely distinct from Aire-expressing mTECs and DCLK1 + thymic tuft cells (Figure 2E).Similar to the postnatal thymus, CCL21-expressing mTECs in the embryonic thymus distributed throughout the medullary region and were not enriched in the pericortical areas including the cortico-medullary junctions (Figure 2F, G, Figure 2-figure supplement 1).These results indicate that CCL21-expressing mTECs arise by E15 in the central region of the embryonic thymus.

Fate mapping of CCL21-expressing cells in thymus
To better understand whether CCL21-expressing mTECs have a developmental potential to give rise to the self-antigen-displaying mTEC subset, we engineered mice in which the P1 bacteriophagederived Cre recombinase gene was knocked-in at Ccl21a locus (Figure 3A).We bred Ccl21a Cre/+ mice with mice that carried a ubiquitous CAG promoter-driven loxP-stop-loxP-EGFP (CAG-loxP-EGFP) transgene.In Ccl21a-Cre × CAG-loxP-EGFP mice, EGFP expression labeled cells that have transcribed Ccl21a previously and/or presently (Figure 3A).
In the quantitative RT-PCR analysis of Cre, Ccl21a, and other TEC-associated genes, such as Aire, Tnfrsf11a, and Psmb11, Cre was detected specifically in the Ccl21a-expressing mTEC low subpopulation, which includes the majority of CCL21-expressing mTECs (Lkhagvasuren et al., 2013), and not in the Psmb11-expressing cTECs or the Aire-and Tnfrsf11a-expressing mTEC high subpopulation (Figure 4A).These results verify the lineage tracing potential of Ccl21a-Cre-expressing cells to differentiate into the mTEC high subpopulation including Aire + mTECs.
In addition to mTECs, approximately 66% of UEA1 − Ly51 + cTECs in the postnatal thymus were EGFP + in 3-week-old Ccl21a-Cre × CAG-loxP-EGFP mice (Figure 3F, G).Section analysis of the thymus verified the presence of EGFP + cells in the cortex (Figure 3B).Because cTECs do not presently express Ccl21a transcripts (Kozai et al., 2017) (also shown in Figure 1B-D), these results indicate that approximately two-thirds of postnatal cTECs are derived from cells that previously transcribed Ccl21a (Figure 3F, G).To characterize cTECs that previously transcribed Ccl21a and those that did not, we isolated EGFP + and EGFP − cTECs from 2-week-old postnatal Ccl21a-Cre × CAG-loxP-EGFP mice (Figure 4-figure supplement 1) and performed deep-sequencing transcriptomic analysis.The MA and volcano plots of RNA-sequencing data demonstrated high similarity in gene expression profiles between EGFP + and EGFP − cTECs among 20,230 genes detected (Figure 4B, C), unlike the pronounced difference in global gene expression profiles between cTECs and mTECs (Figure 4figure supplement 2A, B).The genes that are relevant for T cell-inducing and positive selectioninducing functions in cTECs, including Dll4,Il7,Cxcl12,Psmb11,Ctsl,Prss16,Cd83, were similarly abundant in EGFP + and EGFP − cTECs (Figure 4-figure supplement 2C).These results suggest that cTECs derived from cells that either do or do not transcribe Ccl21a are functionally comparable to promote the generation and positive selection of cortical thymocytes.
Only 14 genes, including Ccl21a, showed significantly higher expression in EGFP + cTECs than in EGFP − cTECs, whereas only one gene, the parathyroid hormone-encoding Pth, showed significantly lower expression in EGFP + cTECs than in EGFP − cTECs (Figure 4C, Figure 4-figure supplement 2D).All the 14 genes that showed significantly higher expression in EGFP + cTECs than in EGFP − cTECs represented genes that showed higher expression in mTECs than in cTECs, and their expression, including Ccl21a expression, was much higher in mTECs than in EGFP + cTECs (Figure 4-figure supplement 2D).Interestingly, EGFP + cTECs distributed unequally within the thymic cortex and were detected more frequently in the perimedullary cortical region than in the central cortical region or the subcapsular region of the cortex (Figure 4D, E, Figure 4-figure supplement 3A).In contrast, Ly51 + total cTECs did not show such an unequal distribution in the thymic cortex (Figure 4-figure supplement 3B, C).Thus, the intrathymic proximity to the medullary region contributes to the previous expression of Ccl21a transcripts in cTECs, suggesting that approximately two-thirds of postnatal cTECs originate from Ccl21a + cells generated in the thymic medulla.
Collectively, these fate-mapping experiments demonstrate that a vast majority of mTECs, including the self-antigen-displaying mTEC subset, and approximately two-thirds of cTECs, particularly cTECs in the perimedullary cortical region, are derived from progenitor cells that previously transcribed Ccl21a.

Ccl21a-expressing mTECs arise early in central region of thymus primordium
It is further interesting to note that EGFP + cells within Foxn1 + mTECs were detected in the central region of the thymus primordium in Ccl21a-Cre × CAG-loxP-EGFP mice as early as E11 (Figure 5A).The detection of EGFP + cells occurred earlier in the ontogeny than the detection of Ccl21a tdTomato+ mTECs by E13 (Figure 2B) and CCL21-protein-expressing mTECs by E15 (Figure 2D).These sequential detections suggested that low-level Ccl21a transcription initiated as early as E11 during thymus organogenesis was sufficient to produce functional Cre proteins to mediate the expression of CAGpromoter-dependent EGFP proteins, but failed to produce a detectable amount of tdTomato or CCL21 proteins.Subsequent elevation of Ccl21a transcription level resulted in the production of detectable amounts of tdTomato proteins followed by CCL21 proteins.Indeed, single-cell RNA-sequencing analysis of Ccl21a transcript counts demonstrated that Ccl21a expression levels within individual Epcam +- Foxn1 + TECs elevated during the embryogenesis from E12 to E14 (Figure 5B).At E14, Ccl21a low cells were detected at an equivalent frequency in β5t + and β5t − TEC populations (Figure 5B).Ccl21a transcription in CCL21-expressing mTECs was further elevated from embryonic to postnatal development, as shown by single-cell RNA-sequencing analysis of Ccl21a transcript counts (Figure 5B) and verified by flow cytometric analysis of Ccl21a-dependent tdTomato expression levels (Figure 5C).Importantly, Ccl21a + Foxn1 + mTECs detected during early thymus organogenesis were localized in the central region of the thymus primordium and were anatomically and transcriptomically distinguishable from Ccl21a + Gcm2 + parathyroid epithelial cells (Figures 2B and 5A,D).These results reveal that Ccl21a + mTECs develop in the central region of the thymus primordium as early as E11 before the generation of detectably CCL21 protein-expressing mTECs in the embryonic thymus by E15.

Developmental potential of CCL21-expressing mTECs
To directly examine whether CCL21-expressing mTECs have a developmental potential to give rise to self-antigen-displaying mTECs, we finally traced the fate of CCL21-expressing mTECs within the thymus microenvironment.To do so, we employed reaggregate thymus organ culture experiments (Anderson et al., 1993;White et al., 2008) where RelB-deficient thymic stromal cells do not generate any functional mTECs (Weih et al., 1995;Burkly et al., 1995).Reaggregate thymuses were transplanted under mouse kidney capsules for reconstitution with hematopoietic cells derived from recipient mice.In this experimental condition, RelB-deficient thymic stromal cells were reaggregated with tdTomato-expressing mTECs highly purified from Ccl21a tdTomato E17 embryos (Figure 8-figure supplement 1).Ccl21a tdTomato+ mTECs had already become functional CCL21-protein-expressing mTECs by E17 (Figure 2), and the accumulation of CD4 + CD8 -and CD4 -CD8 + thymocytes in E17 thymic medullary region was defective in mice deficient in CCL21 (Figure 7A-C), so that the reaggregate thymus experiments would address whether functionally relevant CCL21-expressing mTECs give rise to other mTEC subpopulations including Aire + mTECs.
In contrast to CCL21-expressing mTECs isolated from E17 embryos, Ccl21a tdTomato -positive EpCAM + CD45 − mTECs isolated from 4-week-old Ccl21a tdTomato/+ mice did not exhibit any signs of Aire + Krt14 + mTEC generation in the thymus reaggregated with RelB-deficient thymic stromal cells (Figure 8-figure supplement 2A-G), suggesting a decline in the developmental potential of CCL21 + mTECs during the ontogeny from embryonic to postnatal period.However, no detection of progeny could also result from technical caveats associated with reaggregate thymus organ culture conditions that were suitable for detection of the developmental potential of embryonic but not postnatal CCL21-expressing mTECs, or differences in the lifespan and/or turnover of embryonic and postnatal CCL21-expressing cells.
Throughout the development including E17 embryos, CCL21-expressing mTECs were localized specifically in the central region of the thymus and were Krt14 + (Figures 1, 2, 6, and 9A).However, single-cell RNA-sequencing analysis demonstrated that the global gene expression profiles were largely different between embryonic Ccl21a low mTECs and 4-week-old postnatal Ccl21a high mTECs (Figure 9B).Indeed, the majority of CCL21-expressing mTECs at E17 but not the postnatal period simultaneously expressed cTEC-associated protein Ly51 and mTEC-associated UEA1-binding molecules on the cell surface (Figure 9C).Ly51 expression was lower in embryonic TECs than postnatal TECs (Ripen et al., 2011) and equivalently distributed in the thymus including the medullary region defined by Krt14-expressing mTECs centrally localized in E17 thymus (Figure 9A).These results highlight the difference in molecular expression profiles, including Ly51 expression, between E17 CCL21and postnatal CCL21-expressing mTECs.

Discussion
The present results demonstrate that CCL21-expressing mTECs arise early during embryonic thymus development.Ccl21a-Cre-mediated detection reveals that Ccl21a + Foxn1 + mTECs are detectable as early as E11 in the central region of the embryonic thymus, shortly after the organogenesis of the thymus primordium, and this is followed by the detection of Ccl21a-tdTomato-expressing mTECs by E13 and CCL21 protein-expressing mTECs by E15.Ccl21a-Cre-mediated fate-mapping analysis establishes that at 3 weeks old, a vast majority of mTECs, including the self-antigen-displaying mTECs such as Aire + mTECs and thymic tuft cells, and approximately two-thirds of cTECs originate from Ccl21a + cells.Most importantly, our results demonstrate that Ccl21a-tdTomato-expressing and functional CCL21 protein-producing mTECs isolated from E17 embryonic thymus have a developmental potential to give rise to the self-antigen-displaying mTECs in the thymus microenvironment.These results demonstrate a previously unknown process by which the diversity in the thymus medulla epithelium    Functional conversion of the thymocyte-attracting mTEC subset into the self-antigen-displaying mTEC subset offers an interesting implication that the thymus medulla is formed by initially generating CCL21-expressing mTECs and attracting CCR7-expressing positively selected thymocytes from the cortex even before the subsequent display of self-antigens mediated by Aire + mTECs and thymic   (Kozai et al., 2017;Kurobe et al., 2006;Nitta et al., 2009) underscored the importance of functional CCL21-expressing mTEC subset.Our present results showing developmental potential of functional CCL21-expressing mTECs to give rise to self-antigen-displaying mTECs reveals an additionally important function of CCL21-expressing mTECs in forming the diversity in thymus medulla epithelium, including the self-antigen-displaying mTEC subset.Stepwise progression in mTEC development from thymocyte-attracting function to self-antigen-displaying function fits stepwise requirement to initially recruit newly generated T cells from the thymic cortex before tolerizing T cells to self-antigens.Downregulation of CCL21 in functionally converted self-antigen-displaying mTECs may further contribute to the prevention of excessive negative selection of newly generated T cells to self-antigens.
Recent studies demonstrated that the function of the thymus medulla is not limited to the establishment of self-tolerance in conventional αβ T cells but extends to the development of innate lymphocytes, including invariant NKT cells and γδ T cells (White et al., 2014;Roberts et al., 2012).The developmental capability of CCL21-expressing mTECs may further extend to additional mTEC subpopulations that contribute to supporting the development of innate invariant lymphocytes.Thus, CCL21-expressing mTECs may represent a primordial mTECs which separate newly generated αβ T cells, γδ T cells, and NKT cells from the thymic cortex to the medullary microenvironment for further selection and maturation.
In contrast to the robust generation of Aire + mTECs from embryonic CCL21 + mTECs, our results show that the ability to give rise to Aire + mTECs was not detectable in postnatal CCL21 + mTECs.The fact that no Aire + mTECs were detected in reaggregate thymus organ culture may reflect a postnatal from Ccl21a tdTomato Tnfrsf11a(RANK) Venus mice at indicated embryonic age.(C) Principal component analysis of RNA-sequencing data of indicated cell populations isolated from Ccl21a tdTomato/+ Tnfrsf11a(RANK) Venus E17 embryos.(D) Enrichment analysis of ontology for genes that are differently expressed (RPKM >1, fold change >1.5 or <-1.5, FDR p-value <0.05) between CCL21 + RANK − and CCL21 − RANK + TECs.Bars show the adjusted p-values of the top five categories enriched in CCL21 + RANK − TECs (top) and CCL21 − RANK + TECs (bottom).Numbers in parentheses indicate the number of categorized genes.(E) RPKM values of indicated genes detected in RNA-sequencing analysis.
The online version of this article includes the following figure supplement(s) for figure 6:   decline in the developmental potential of CCL21 + mTECs.However, it is also possible that the developmental kinetics and the requirements for postnatal CCL21 + mTECs may be different from those for E17 CCL21 + mTECs, so that the reaggregate thymus organ culture conditions for detecting the development of E17 CCL21 + mTECs may not be optimal for the detection of the survival and development of postnatal CCL21 + mTECs.
The online version of this article includes the following figure supplement(s) for figure 8:   Nevertheless, it is important to notice the difference in the developmental potential between embryonic and postnatal CCL21 + mTECs in reaggregate thymus organ culture.Analysis of molecular expression profiles further highlighted the difference between embryonic and postnatal CCL21 + mTECs.We think that CCL21 + mTECs may be heterogenous in developmental capability.Embryonic CCL21 + mTECs, which give rise to self-antigen-displaying mTECs including Aire + mTECs and mimetic mTECs, may also terminally differentiate into a subpopulation of CCL21 + mTECs, which no longer have further developmental potential, and which represent the majority of postnatal CCL21 + mTECs.
Interestingly, our results also reveal that approximately two-thirds of cTECs are derived from progenitor cells that previously transcribed Ccl21a.The previous expression of Ccl21a transcripts in the fraction of cTECs is influenced by the intrathymic proximity to the medullary region and is enriched in the perimedullary cortical region (Figure 4E).Developmental capability to become cTECs is shown in Ccl21a gene-expressing cells, and it is unknown whether CCL21-proteinexpressing functional mTECs retain the developmental potential to give rise to cTECs.In contrast, our results from reaggregate thymus experiments clarified that CCL21-protein-expressing functional mTECs have a developmental potential to convert into Aire-expressing mTECs.Consequently, we think that at least a fraction of Ccl21a transcript-expressing mTECs retain a bipotent developmental potential to give rise to both cTECs and mTECs.It is unclear whether most Ccl21aexpressing mTECs retain a bipotent developmental potential for cTECs and mTECs or Ccl21aexpressing mTECs represent a mixture of bipotent progenitors and lineage-committed mTEC progenitors.Because of the unequal distribution of EGFP-labeled cTECs within the thymic cortex, initial signals to promote bipotent progenitors to mTEC lineage may be provided by molecules restricted in the central region of the embryonic thymus, and those signals may promote the transcription of Ccl21a at an early stage of mTECs, which still retain the potential to become cTECs.Further examination of cTEC potential, for example, by using CCL21-tdTomato-expressing cells that constitutively express GFP, may lead to a better understanding of the cTEC-generating capability in embryonic and postnatal CCL21 + mTECs.It is also interesting to examine whether GFP + cTEC development in Ccl21a-Cre × CAG-loxP-EGFP mice is mediated through RelB-dependent mTEC developmental progression and/or dependent on developing thymocyte-dependent mTECnurturing 'crosstalk' signals.Nusser et al., 2022 recently identified two bipotent TEC progenitor populations: an early cTECbiased progenitor population and a postnatal mTEC-biased progenitor population.Interestingly, they noted that the postnatally appearing mTEC-biased progenitor population includes cells that transcribe Ccl21a (Nusser et al., 2022).It would be interesting to clarify whether and how those postnatal TEC progenitors overlap with the embryonic and postnatal CCL21-protein-expressing mTECs reported in this study.It would also be interesting to shed light on how differently Ccl21a + progenitors contribute to cTECs and mTECs over the ontogeny and whether the enrichment of Ccl21a + progenitor-derived cTECs in the perimedullary area reflects a temporal replacement of cTECs derived from Ccl21a + progenitors localized in the medulla.
Further characterization of CCL21-expressing mTECs carrying a developmental potential to give rise to self-antigen-displaying mTEC subset is of great interest.Our results highlight the difference between CCL21-expressing mTECs and previously characterized RANK-expressing mTEC-restricted progenitors.CCL21-and RANK-expressing mTECs are detectable in E13 embryonic thymus, are proximally localized within the medullary region in the thymus, and are capable of generating Aireexpressing mTECs.Unlike RANK-expressing mTECs, however, CCL21-expressing mTECs include developmental potential to give rise to cTECs.Gene expression profiles show that CCL21-expressing mTECs retained more cTEC-trait genes, including Psmb11, Cxcl12, CD83, and Ackr4, than RANKexpressing mTECs, suggesting that in comparison with RANK-expressing mTECs, CCL21-expressing mTECs are more closely related to and more recently derived from cTEC-trait-expressing bipotent TEC progenitors.Furthermore, it is interesting to note that recently described Krt19 + progenitors that give rise to multiple mature mTEC populations including Aire + mTECs, mimetic mTECs, and postnatal CCL21 + mTECs, lack expression of CCL21 and RANK (Lucas et al., 2023).An important aspect of future work will be to examine whether CCL21-, RANK-, and Krt19-expressing mTECs are linked in upstream-downstream relationships in a single developmental pathway, or are developmentally distinct and parallel progenitors to give rise to mTECs.Similarly, it will be interesting to better clarify possible heterogeneity in CCL21-expressing mTECs in relation to their ability to give rise to cTECs as well as mTECs.Nevertheless, the finding in our study that CCL21 + embryonic mTECs can give rise to Aire + mTECs reveals a new source of an important component of the thymus medulla.
Our results show that 95% of mTECs are labeled with EGFP fluorescence and therefore derived from Ccl21a + progenitors, whereas 5% of mTECs are not labeled with EGFP fluorescence in Ccl21a-Cre × loxP-EGFP mice.Our previous study using β5t-driven Cre mice indicated fate-mapping of 95% EGFP + mTECs in CAG-loxP-stop-loxP-EGFP-transgenic reporter mice used in this study and >99.5% ZsGreen + mTECs in Rosa26-loxP-ZsGreen-knock-in reporter mice (Ohigashi et al., 2013).Consequently, we think that 95% EGFP + cells in mTECs detected in this study may indicate that virtually all (>99.5%)mTECs are derived from cells that once transcribed Ccl21a.
A previous study detected Ccl21a transcripts within a podoplanin-expressing TEC subpopulation that resembled immature TECs in postnatal thymus (Onder et al., 2015).It was reported that podoplanin-expressing mTEC progenitors, also known as junctional TECs (jTECs), were localized at the cortico-medullary junction (Onder et al., 2015).However, our results show that CCL21-expressing mTECs in the embryonic and postnatal thymus distribute throughout the medullary region and are not enriched in the cortico-medullary junctions (Figures 1H and 2G).Indeed, the majority of CCL21expressing mTECs in postnatal thymus did not express podoplanin (Figure 8-figure supplement 2H).Moreover, podoplanin expression was broadly detected by the majority of embryonic E17 TECs, most of which were cTECs (Figure 8-figure supplement 2H).Thus, neither podoplanin expression nor cortico-medullary junctional distribution characterizes CCL21-expressing mTECs.More importantly, our results demonstrate developmental potential in thymocyte-attracting CCL21-expressing mTECs and identify developmental conversion between functionally distinct mTEC subsets, which serves to assemble a diverse medullary microenvironment in the thymus.
In conclusion, the present study establishes that CCL21-expressing functional mTEC subset carries a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of functionally distinct mTEC subsets from thymocyte-attracting cells into self-antigendisplaying cells contributes to the functional diversification of thymus medulla epithelium during embryonic development.The present results are likely to ignite many interesting questions, including the characterization of signals that drive the developmental conversion of mTECs and the elucidation of developmental pathways for diverse TEC subpopulations.

Reaggregate thymus experiments
Fetal thymus lobes isolated from RelB-KO mice at embryonic day (E) 15 were organ cultured for 5 days on sponge-supported Nucleopore filters (Whatman) placed on Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and penicillin-streptomycin mixed solution.To eliminate endogenous hematopoietic cells from the lobes, 1.35 mM 2′-deoxyguanosine (dGuo) was added in the culture medium (Anderson et al., 1993;White et al., 2008).dGuo-treated thymus lobes were digested with 0.25% trypsin supplemented with 0.45 mM EDTA (ethylenediaminetetraacetic acid) and 0.02% DNase I for 15 min at 37 °C and reaggregated with tdTomato + or tdTomato − TECs isolated from Ccl21a tdTomato/+ mice at E17 or 4 weeks old.After 1-day culture on Nucleopore filters, reaggregated thymuses were transplanted under kidney capsules of B6 mice.dGuo-treated fetal thymus lobes isolated from RelB-deficient and RelB-heterozygous mice were also transplanted under kidney capsules of B6 mice.Grafts were harvested 5 weeks post transplantation.

Fluorescence distribution analysis
The plot profile intensity for the fluorescence image of each channel in the region of interest (ROI) was measured using ImageJ software (https://imagej.nih.gov/ij/index.html).The fluorescence intensity at each horizontal distance with the total intensity of the vertically averaged tdTomato, EGFP, or Ly51 in the ROI was calculated as 100.The sum of tdTomato or EGFP intensities for the medullary region defined by Krt14 expression or UEA1 reactivity and for the cortical region was calculated.

RNA-sequencing analysis
cDNAs were prepared by using SMART-Seq v4 Ultra Low Input RNA Kit, according to the manufacturer's protocol (Clontech).Sequencing libraries were generated by using a Nextera XT DNA Library Prep Kit, according to the manufacturer's protocol (Illumina).The concentration of libraries was measured by an ABI PRISM 7500 Real-time PCR system in combination with a Power SYBR Green PCR Master Mix (Thermo Fisher).Single-end sequencing of cDNA libraries with a read length of 51 was performed with HiSeq 1500 platform (Illumina).For RNA-sequencing analysis of CCL21 + RANK − and CCL21 − RANK + TECs isolated from Ccl21a tdTomato Tnfrsf11a(RANK) Venus embryonic E17 thymus, RNA was prepared using QIAGEN RNAeasy Plus Micro Kit with genomic DNA eliminator columns.RNAsequencing samples were sequenced on NovaSeq_SP flowcell using Illumina TruSeq Stranded Total RNA Library Prep with 2 × 100 bp paired-end sequencing.Data were analyzed by using CLC Genomics Workbench 12.0 (QIAGEN) with default parameters.
Single-cell RNA-sequencing analysis E12 embryonic pharyngeal cells, E14 embryonic CD45 − EpCAM + β5t-Venus + and β5t-Venus − TECs, E16 embryonic CD45 − EpCAM + β5t-Venus + and β5t-Venus − TECs isolated from β5t-Venus knock-in mice, and 4-week-old postnatal TEC-enriched thymus cells were employed for single-cell RNA-sequencing.3′ mRNA-seq library was generated as outlined in the Chromium Connect platform using 3' v3.1 Single Cell RNA-Seq chemistry reagents from 10× Genomics.Reverse transcription and cDNA amplification were performed according to the manufacturer's instructions.Libraries were sequenced on NextSeq 2000 and FASTQ files were generated from demultiplexes raw base call files using Cell Ranger 6.1.1 mkfastq.Alignment, filtering, barcode counting, and UMI (unique molecular identifier) counting were performed using Cell Ranger 6.1.1 count with mouse reference 3.0.0(mm10 and Ensembl release 98).The resulting matrix was imported into Seurat (v4.3.0) in R v 4.3.0 to create a Seurat object.The object underwent normalization, filtering, scaling, and dimensionality reduction steps sequentially using Seurat build-in functions, with default parameters.To integrate all the filtered objects from each sample, the CCA algorithm was employed through the FindIntegrationAnchors and IntegrateData functions in Seurat.The Uniform Manifold Approximation and Projection plots were visualized using the DimPlot function of Seurat.The histograms were visualized using the ggplot2 package (v3.4.2).

Statistical analysis
Statistical significance was assessed using the two-tailed unpaired Student's t-test with Welch's correction for unequal variances or one-way analysis of variance with Tukey's correction.

F)
Figure supplement 1. Distribution of Ccl21a-expressing cells in postnatal thymus.

Figure 2 .
Figure 2. CCL21-expressing medullary thymic epithelial cells (mTECs) in embryonic thymus.(A) tdTomato fluorescence (white) detected in thymus sections from E15 Ccl21a tdTomato/+ embryos.White lines indicate the capsular outline of the thymus.The image in central region identified by the box in upper panel is magnified in bottom panel.Representative data from at least three independent experiments are shown.Scale bar, 200 μm.(B) tdTomato (red) and GFP (green fluorescence protein) (green) fluorescence in thymus sections from E13 Ccl21a tdTomato/+ × Foxn1-GFP-transgenic embryos (left).Dashed lines show the outlines for the thymus and parathyroid primordia.Central region of the thymus identified by the box in upper panel is magnified in bottom panel.Scale bar, 100 μm.Right panel shows a representative flow cytometric profile for tdTomato and GFP in CD45 − EpCAM + epithelial Figure 2 continued on next page Figure supplement 1. Distribution of Ccl21a-expressing cells in embryonic thymus.

Figure supplement 3 .
Figure supplement 3. Distribution of EGFP + cells in the thymus.

Figure 4
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Figure 6 .
Figure 6.CCL21-expressing medullary thymic epithelial cells (mTECs) are distinct from RANK-expressing mTECs.(A) Immunofluorescence analysis of tdTomato (red) and Venus (green) signals in thymus sections from Ccl21a tdTomato/+ Tnfrsf11a(RANK) Venus mice at indicated embryonic age.White lines indicate the capsular outline of the thymus.Images in boxed regions in upper panels are magnified in bottom panels.Representative data from at least three independent experiments are shown.(B) Flow cytometric profiles of tdTomato and fluorescence signals in EpCAM + CD45 − TECs isolated Figure 6 continued on next page

Figure supplement 1 .
Figure supplement 1. Purity of isolated TECs for transcriptomic analysis.

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Figure 8 .
Figure 8. Developmental potential of CCL21-expressing medullary thymic epithelial cells (mTECs).(A-D) Immunofluorescent staining of indicated thymus graft sections.Shown in upper panels are Krt14 expression (white).White lines indicate capsular outline of the thymus.Blue lines show Krt14 + medullary region.Scale bar, 500 μm.Bottom panels show Krt14 (white) and Aire (magenta) fluorescence signals within yellow boxes in upper panels.Scale bar, 50 μm.Grafts represent RelB-KO thymic stroma reaggregated with tdTomato + TECs (A) and tdTomato − TECs (B) as well as fetal thymus lobe from RelB-KO mice (C) and RelB-heterozygous mice (D).(E) Size of Krt14 + medullary areas (means and standard error of the means [SEMs], n = 6) in Figure 8 continued on next page

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Figure 8 continued