Hematopoietic lymphoid tissue inducer (LTi) cells are essential for the development of secondary lymphoid tissues including lymph nodes and Peyer’s patches. Two transcription factors, the helix-loop-helix inhibitor Id2 and the retinoic acid-related orphan receptor γt (Rorγt), have been shown to be crucial for LTi cell development. However, it remains unclear how the specification of multipotent hematopoietic progenitor cells toward the LTi lineage is programmed. In this study, we report impaired lymphoid tissue organogenesis in mice in which the function of Runx1/Cbfβ transcription factor complexes was attenuated by the loss of either the distal promoter-derived Runx1 or Cbfβ2 variant protein. We found that LTi progenitors in fetal liver, defined previously as a lineage marker-negative α4β7 integrin (α4β7)+ IL-7R α-chain (IL-7Rα)+ population, can be subdivided into Rorγt-expressing IL-7Rαhigh cells and nonexpressing IL-7Rαmid cells. Whereas Id2 and Rorγt are required to direct α4β7+IL-7Rαmid cells to become α4β7+IL-7Rαhigh cells, Runx1/Cbfβ2 complexes are necessary for the emergence of α4β7+IL-7Rαmid cells. In addition, the loss of Cbfβ2, but not P1-Runx1, resulted in an inefficient upregulation of Rorγt in residual α4β7+IL-7Rα+ LTi cells at anlagen. Our results thus revealed that Runx1/Cbfβ2 complexes regulate the differentiation of LTi cells at two stages: an early specification of hematopoietic progenitors toward the LTi lineage and a subsequent activation of Rorγt expression at anlagen.

The development of the immune system involves the differentiation of effecter cells and lymphoid organogenesis. Peripheral secondary lymphoid tissues, such as lymph nodes (LNs) and Peyer’s patches (PPs), are essential sites at which the immune responses take place and are regulated. The formation of anlagen of these tissues is initiated during embryogenesis via the interaction of hematopoietic lymphoid tissue inducer (LTi) cells with mesenchymal lymphoid tissue organizer (LTo) cells (1, 2). Although previous studies have provided molecular insights into how LTi cells migrate correctly and communicate with LTo cells to initiate anlagen formation (35), the early differentiation pathway of LTi cells from multipotent hematopoietic progenitor cells remains to be characterized.

CD4+CD3 cells that accumulate in neonatal lymph nodes were first described by Kelly and Scollay (6) and are found as cell clusters in the intestines of 17.5 d postconception (dpc) embryos (7). These CD4+CD3 cells express molecules involved in lymphoid organogenesis such as lymphotoxin-β and chemokine receptor CXCR5 (8). Adoptive transfer of a CD4+CD3 population isolated from fetal spleen or intestine has shown that these cells are responsible for lymphoid tissue formation (5, 9). Several other surface markers were shown to be useful to trace the differentiation pathway of LTi cells. For instance, expression of the IL-7R α-chain (IL-7Rα) enables us to identify LTi progenitors in lineage marker-negative (Lin) populations (10, 11). In addition, coexpression of α4β7integrin (α4β7) with IL-7Rα in CD4+CD3 cells at neonatal LN suggested that α4β7 is another marker useful for defining LTi cells (8, 12, 13). Interestingly, a Linα4β7+IL-7Rα+ population is present in 12.5 dpc fetal liver cells (13). Reconstitution of CD4+CD3 population in vivo by injection of IL-7Rα+ fetal liver cells suggests that LinIL-7Rα+ fetal liver populations are likely to contain precursors of LTi cells (14). In addition, by employing an in vitro culture system, Linα4β7IL-7Rα+ fetal liver population, in which B-lymphoid potential is also retained, was shown to give rise to α4β7+IL-7Rα+ cells that lacked B-lymphoid potential (13). These results suggest that the early specification to the LTi lineage takes place in Linα4β7IL-7Rα+ population in the fetal liver.

In contrast, genetic approaches have shown that both the retinoic acid-related orphan receptor γt (Rorγt) and the helix-loop-helix inhibitor Id2 are essential for the differentiation of LTi cells (15, 16). Rorγt is encoded by the Rorc gene, which produces two isoforms, Rorγ and Rorγt, from distinct promoters (17). Expression of Rorγt from the proximal promoter is restricted to LTi cells and some T-lymphocyte subsets (18). Accumulation of CD4+CD3 cells in the fetal intestine was abrogated in Rorγt-deficient mice (18), indicating that induction of Rorγt expression via activating the Rorγt promoter is essential for the development of LTi cells. To further understand the differentiation pathway of LTi cells, it is therefore important to understand the mechanism regulating Rorγt promoter activity and to clarify the developmental stage at which Rorγt and Id2 function to regulate LTi cell differentiation.

Runx complexes are evolutionary conserved heterodimeric transcriptional regulators that have been shown to be essential for differentiation of several hematopoietic lineage cells, including hematopoietic stem cells, B-lymphocytes, NKT cells, and cytotoxic T cells (1922). Runx complexes are composed of a DNA-binding Runx protein and its non–DNA-binding partner Cbfβ (Supplemental Fig. 1). All three mammalian Runx genes (Runx1, Runx2, and Runx3) are transcribed from the distal (P1) and proximal (P2) promoters (23). In addition to the differential expression pattern of P1- and P2-derived Runx1 variants (P1-Runx1 and P2-Runx1, respectively) (24), P1-Runx1 and P2-Runx1 variants differ in their N-terminal end sequences (Supplemental Fig. 1). Conversely, alternative RNA splicing of the Cbfβ transcript generates two Cbfβ splicing variants, Cbfβ1 and Cbfβ2, which differ only at their C termini (Supplemental Fig. 1) (25).

In this study, we report that mice lacking either the P1-Runx1 or Cbfβ2 variant exhibit impaired lymphoid tissue organogenesis due to the impaired differentiation of LTi cells. Our results demonstrate that a Linα4β7+IL-7Rα+ population in the fetal liver can be divided into RorγtIL-7Rαmid and Rorγt+IL-7Rαhigh subsets. Interestingly, loss of P1-Runx1 and Cbfβ2, but not Rorγt nor Id2, results in the severely impaired differentiation of Linα4β7+IL-7Rαmid cells, indicating that Runx1/Cbfβ2 complexes are involved in the early specification to the LTi lineage prior to the developmental stage at which Rorγt and Id2 further program LTi cell development. In addition, full induction of Rorγt expression at anlagen is dependent on Cbfβ2. These results reveal two Runx1/Cbfβ2-dependent regulatory mechanisms underlying LTi cell development: an early specification toward the LTi lineage in multipotent progenitors and an involvement in an induction of Rorγt expression later at anlagen.

Rorγtgfp/gfp, Id2−/−, and Runx1PIN/P1N mice have been described previously (15, 18, 26). The generation of Cbfβ2m/2m mice by homologous recombination in embryonic stem cells will be described elsewhere. In brief, to generate the Cbfβ2m allele, the splice donor signal, the GTTAG sequences, at the end of the exon 5 in the Cbfβ gene, was mutated to AATTC. Because the targeted mutations inhibited RNA splicing generating Cbfβ2 mRNA, no Cbfβ2 variant protein is produced in the Cbfβ2m/2m mice.

Whole-mount immunostaining was performed as previously described (7). In brief, excised fetuses were microwave irradiated at 500 W for 30 s in ice-cooled fixing solution (2% paraformaldehyde in PBS) and incubated in the same solution for 30 min at 4°C. Adult small intestine was fixed in 4% paraformaldehyde in PBS for 2 h at 4°C. The specimens were dehydrated by incubating them for 30 min each with 50, 75, 90, and 100% methanol in PBS at 4°C. After removing the serosa of the adult small intestine, the endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 30 min at 4°C. The rehydrated specimens were then stained with anti-B220, anti–IL-7Rα, or anti–VCAM-1 mAb, which was visualized with an HRP-conjugated secondary Ab.

The Abs used for flow cytometry were from BD Pharmingen or eBioscience: CD3ε (clone 145-2C11), CD11c (HL3), CD45R/B220 (RA3-6B2), NK-1.1 (PK136), Gr-1 (RB3-8C5), TER-119 (TER-119), α4β7 (DATK32), IL-7Rα (A7R34), CD16/CD32 (2.4G2), c-Kit (2B8), Sca-1 (E13-161.7), CD4 (L3T4), CD8α (Ly-2), CXCR5 (2G8), and TCRβ (H57-597). Surface staining was performed for 15–20 min with the corresponding mixture of fluorescently labeled Abs. Data were acquired on an FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

All organs of embryos were dissected under a stereomicroscope and then dissociated with dispase (BD Pharmingen) for 15 min at 37°C, as previously described (10, 13). After gently pipetting, the dissociated cells were washed with PBS containing 2% FCS. When staining for CXCR5 and CD4, the organs were dissociated with collagenase (Wako Chemicals).

To investigate the function of P1-Runx1 variant protein, we replaced the exon encoding-specific N-terminal sequences for P1-Runx1 with the neor gene (the Runx1P1N allele) (26). Although NKT cell development was almost abolished in the Runx1P1N/P1N mice, as reflected in the conditional inactivation of the Runx1 gene in CD4+CD8+ double-positive thymocytes (21), other phenotypes, such as thymic hypocellularity, CD4 derepression in double-negative thymocytes, and the reduction of CD4+ T cell numbers, were milder than those caused by inactivation of Runx1 gene (Supplemental Fig. 2) (27, 28). Thus, the effect of P1-Runx1 deficiency varied among T cell subsets and the developmental stages, presumably because of differences in either a redundant function of the P2-Runx1 protein or in the sensitivity to the dosage of Runx1 protein.

During phenotypic analyses of T cell development, we noticed that the formation of PPs was impaired in the Runx1P1N/P1N mice. Immune staining of whole adult intestines with anti-B220 Ab confirmed that both the number and size of PPs were reduced in the Runx1P1N/P1N mice (Fig. 1A–C). Similarly, formation of peripheral LNs, such as the inguinal and axillary LNs, was impaired in the Runx1P1N/P1N mice, whereas the mesenteric LNs were present in all Runx1P1N/P1N mice examined (Fig. 1D).

FIGURE 1.

Impaired development of secondary lymphoid tissues in Runx1P1N/P1N and Cbfβ2m/2m mice. A, Representative gross image of PPs on adult intestine from 12-wk-old control (Wt), Runx1P1N/P1N, and Cbfβ2m/2m mice showing reduced size of remaining PPs in Runx1P1N/P1N and Cbfβ2m/2m mice. Scale bars, 1 mm. B, Whole-mount staining of adult intestine from Wt, Runx1P1N/P1N, Cbfβ+/2m and Cbfβ2m/2m mice using anti-B220 Ab. C, Statistical summary of the numbers of PPs in the littermate control, Runx1P1N/P1N, and Cbfβ2m/2m mice. D, The numerator indicates the number of mice with normal lymph nodes at the indicated positions. Although the mesenteric LNs were present in all Runx1P1N/P1N and Cbfβ2m/2m mice, the development of other peripheral LNs was impaired in these mice. ***p < 0.0001.

FIGURE 1.

Impaired development of secondary lymphoid tissues in Runx1P1N/P1N and Cbfβ2m/2m mice. A, Representative gross image of PPs on adult intestine from 12-wk-old control (Wt), Runx1P1N/P1N, and Cbfβ2m/2m mice showing reduced size of remaining PPs in Runx1P1N/P1N and Cbfβ2m/2m mice. Scale bars, 1 mm. B, Whole-mount staining of adult intestine from Wt, Runx1P1N/P1N, Cbfβ+/2m and Cbfβ2m/2m mice using anti-B220 Ab. C, Statistical summary of the numbers of PPs in the littermate control, Runx1P1N/P1N, and Cbfβ2m/2m mice. D, The numerator indicates the number of mice with normal lymph nodes at the indicated positions. Although the mesenteric LNs were present in all Runx1P1N/P1N and Cbfβ2m/2m mice, the development of other peripheral LNs was impaired in these mice. ***p < 0.0001.

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Because the activity of Runx1 protein is exerted as a heterodimeric complex with a non-DNA partner, the Cbfβ protein (29), we wished to examine the role of Cbfβ in lymphoid tissue organogenesis. By targeting mutations to the splicing donor signals that generate transcript encoding the Cbfβ1 and Cbfβ2 variants, we recently generated Cbfβ1m/1m and Cbfβ2m/2m mice that specifically lack the Cbfβ1 or Cbfβ2 variant, respectively (M. Tenno, Y. Naoe, K. Akiyama, S. Muroi, T. Yasuda, H. Yoshida, and I. Taniuchi, submitted for publication). Whereas Cbfβ1m/1m mice had a normal number of PPs (M. Tachibana, unpublished observations), half of the Cbfβ2m/2m mice lacked PPs formation, and the other half of them contained fewer than four PPs (Fig. 1A–C). Furthermore, formation of peripheral LNs was severely impaired in the Cbfβ2m/2m mice (Fig. 1D), although mesenteric LNs were present in all Cbfβ2m/2m mice.

To examine lymphoid tissue anlagen formation during embryogenesis, whole-mount immune staining of tissues from newborn mice, which enabled us to visualize PP and LN anlagen as an aggregation of LTi or LTo cells, was performed (7). Whereas 6–10 dense-brown signals, representing aggregates of VCAM-1+ LTo cells, were detected on the intestinal wall of the newborn control mice, only a few small, light brown signals were observed in most of the newborn Runx1P1N/P1N mice (Fig. 2A). In the Cbfβ2m/2m fetuses, LTo cell aggregates were either undetectable or barely detected (Fig. 2A). Although counterstaining with anti–IL-7Rα Ab easily detected LTi cell aggregates in control mice, reduced numbers of IL-7R+ cells were detected in a relatively scattered manner in Runx1P1N/P1N and Cbfβ2m/2m mice (Fig. 2B). Similarly, although LN anlagens were detected as an aggregates of IL-7Rα+ LTi cells in the para-aortic regions of the control mice as paired dense spots, a reduced number of diffuse spots or no significant signal was detected in the Runx1P1N/P1N and Cbfβ2m/2m mice (Fig. 2C). Ectopic anlagen formation was occasionally observed in these mutant mice, consistent with the ectopic locations of para-aortic LNs in some adult mutant mice (M. Tachibana and H. Yoshida, unpublished observations). Consistent with the presence of MLN in adult Cbfβ2m/2m mice, formation of MLN anlagen was not impaired by loss of the Cbfβ2 variant (Fig. 2D). These results revealed a novel and essential function of Runx1/Cbfβ2 complexes in the development of the lymphoid tissues anlagen during embryogenesis.

FIGURE 2.

Impaired organization of secondary lymphoid tissue anlagen at 17.5 dpc fetuses of Runx1P1N/P1N and Cbfβ2m/2m mice. Whole-mount staining of intestine with anti–VCAM-1 Ab (A) and anti–IL-7Rα Ab (B) and staining of para-aortic region with anti–IL-7Rα Ab (C) from control (Wt), Runx1P1N/P1N, and Cbfβ2m/2m newborn mice. The brown spot indicated with the arrows in A and C represent clusters of LTo cells at PP anlagen and clusters of LTi cells at the renal or para-aortic LN anlagen, respectively. The blue spot indicated with arrows in left three panels in B represents aggregates of IL-7Rα+ LTi cells at PP anlagen. Images with large magnification indicate a relatively scattered distribution of reduced number of IL-7Rα+ LTi cells in the Cbfβ2m/2m mice (B, right two panels; scale bars, 100 μm). In both Runx1P1N/P1N and Cbfβ2m/2m mice, the numbers of PP and LN anlagen were reduced. The positions of the residual para-aortic LN anlagen, shown by the arrows, were sometimes ectopic in the Runx1P1N/P1N specimen. D, Whole-mount staining of the mesenterium from Cbfβ2m/2m newborn mice with anti–VCAM-1 Ab. The dark blue spots indicated with arrowheads represent the mesenteric LN (MLN) anlagen. Consistent with the presence of MLN in adult Cbfβ2m/2m mice, formation of MLN anlagen was not impaired by loss of Cbfβ2 variant.

FIGURE 2.

Impaired organization of secondary lymphoid tissue anlagen at 17.5 dpc fetuses of Runx1P1N/P1N and Cbfβ2m/2m mice. Whole-mount staining of intestine with anti–VCAM-1 Ab (A) and anti–IL-7Rα Ab (B) and staining of para-aortic region with anti–IL-7Rα Ab (C) from control (Wt), Runx1P1N/P1N, and Cbfβ2m/2m newborn mice. The brown spot indicated with the arrows in A and C represent clusters of LTo cells at PP anlagen and clusters of LTi cells at the renal or para-aortic LN anlagen, respectively. The blue spot indicated with arrows in left three panels in B represents aggregates of IL-7Rα+ LTi cells at PP anlagen. Images with large magnification indicate a relatively scattered distribution of reduced number of IL-7Rα+ LTi cells in the Cbfβ2m/2m mice (B, right two panels; scale bars, 100 μm). In both Runx1P1N/P1N and Cbfβ2m/2m mice, the numbers of PP and LN anlagen were reduced. The positions of the residual para-aortic LN anlagen, shown by the arrows, were sometimes ectopic in the Runx1P1N/P1N specimen. D, Whole-mount staining of the mesenterium from Cbfβ2m/2m newborn mice with anti–VCAM-1 Ab. The dark blue spots indicated with arrowheads represent the mesenteric LN (MLN) anlagen. Consistent with the presence of MLN in adult Cbfβ2m/2m mice, formation of MLN anlagen was not impaired by loss of Cbfβ2 variant.

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To further examine whether a scattered distribution of IL-7R+ cells is caused by a migratory defect or by a reduced number of LTi cells, we performed a flow cytometry analysis of 17.5 dpc fetal intestines. Whereas LTi cells were detected in Lin (CD3, B220, CD11c, NK1.1, Gr-1, and Ter-119) CD45+ population as α4β7+IL-7R+ cells in the control mice, this cell subset was almost absent in the Rorγt-deficient (RorγtGFP/GFP) fetuses (Fig. 3A), as previously reported in histological analyses (18). In Runx1P1N/P1N and Cbfβ2m/2m fetuses, the proportions of Linα4β7+IL-7Rα+ cells were significantly reduced (Fig. 3A). In contrast, the expression level of CXCR5, which is required for the migration of LTi precursors to the appropriate place to initiate anlagen formation (30), was unaffected in the residual Linα4β7+IL-7Rα+ cells by the loss of either the P1-Runx1 or Cbfβ2 variant (Fig. 3B). This suggests that an impairment in differentiation, rather than in migration, of LTi cells is most likely responsible for the resulting impairment in anlagen formation.

FIGURE 3.

Impaired differentiation of LTi at PP anlagen in Runx1P1N/P1N and Cbfβ2m/2m fetuses. A, Expression profiles of α4β7 and IL-7Rα in Lin CD45+ gated fetal intestinal cells from 17.5 dpc fetuses, showing the impaired differentiation of α4β7+IL-7Rα+ LTi cells in Runx1P1N/P1N, Cbfβ2m/2m, and Rorγt-deficient Rorγtgfp/gfp fetuses. B, CXCR5 expression on Linα4β7+IL-7Rα+ fetal intestine cells from control (thin line) and Cbfβ2m/2m (bold line) 17.5 dpc embryos. The shaded histogram represents staining with an isotype control Ab. Data shown are representative of two experiments. C, GFP expression from the Rorγtgfp allele in the Linα4β7+IL-7Rα+ population in 17.5 dpc fetal intestines. The shaded histogram represents data from mice negative for the Rorγtgfp allele. The thin line, the dotted line, and the bold line are from control, Runx1P1N/P1N, and Cbfβ2m/2m fetuses harboring one Rorγtgfp allele, respectively. D, Expression profiles of CD4 and IL-7Rα in the Linα4β7+IL-7Rα+ populations in embryonic day 17.5 fetal intestines. The Linα4β7+IL-7Rα+ population was divided into GFPlow and GFPhigh populations in the control Rorγt+/gfp mice. CD4 expression correlated well with induction of GFP expression. CD4 upregulation was observed in Linα4β7+IL-7Rα+ cells expressing low GFP in Cbfβ2m/2m fetuses. Data shown are representative of two experiments. E, GFP expression from the Rorγtgfp allele in Linα4β7+IL-7Rα+ cells prepared from adult intestines is shown as in C. The data are representative of five experiments.

FIGURE 3.

Impaired differentiation of LTi at PP anlagen in Runx1P1N/P1N and Cbfβ2m/2m fetuses. A, Expression profiles of α4β7 and IL-7Rα in Lin CD45+ gated fetal intestinal cells from 17.5 dpc fetuses, showing the impaired differentiation of α4β7+IL-7Rα+ LTi cells in Runx1P1N/P1N, Cbfβ2m/2m, and Rorγt-deficient Rorγtgfp/gfp fetuses. B, CXCR5 expression on Linα4β7+IL-7Rα+ fetal intestine cells from control (thin line) and Cbfβ2m/2m (bold line) 17.5 dpc embryos. The shaded histogram represents staining with an isotype control Ab. Data shown are representative of two experiments. C, GFP expression from the Rorγtgfp allele in the Linα4β7+IL-7Rα+ population in 17.5 dpc fetal intestines. The shaded histogram represents data from mice negative for the Rorγtgfp allele. The thin line, the dotted line, and the bold line are from control, Runx1P1N/P1N, and Cbfβ2m/2m fetuses harboring one Rorγtgfp allele, respectively. D, Expression profiles of CD4 and IL-7Rα in the Linα4β7+IL-7Rα+ populations in embryonic day 17.5 fetal intestines. The Linα4β7+IL-7Rα+ population was divided into GFPlow and GFPhigh populations in the control Rorγt+/gfp mice. CD4 expression correlated well with induction of GFP expression. CD4 upregulation was observed in Linα4β7+IL-7Rα+ cells expressing low GFP in Cbfβ2m/2m fetuses. Data shown are representative of two experiments. E, GFP expression from the Rorγtgfp allele in Linα4β7+IL-7Rα+ cells prepared from adult intestines is shown as in C. The data are representative of five experiments.

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Because GFP expression from the Rorγtgfp allele is a powerful genetic tool to mark LTi-lineage cells in embryos (18), we introduced the Rorγtgfp allele onto a Runx1P1N/P1N or Cbfβ2m/2m background. Interestingly, GFP expression levels in Linα4β7+IL-7Rα+ cells were lower in the Cbfβ2m/2m fetuses than in the control or Runx1P1N/P1N fetuses (Fig. 3C). This observation can be explained either by developmental inhibition at the transition from the Rorγtlow to the Rorγthigh stage or by an inefficient upregulation of the Rorγt gene. To distinguish between these two possibilities, we examined other markers that might define the developmental stage of LTi cells at anlagen. Upregulation of CD4 expression in Linα4β7+IL-7Rα+ cells was accompanied by the upregulation of GFP from the Rorγtgfp locus in the control fetuses (Fig. 3D). Thus, there are two cell subsets, RorγtmidCD4lo/− and RorγthighCD4+, in the Linα4β7+IL-7Rα+ population at anlagen. In the Cbfβ2m/2m fetuses, CD4 expression levels in Linα4β7+IL-7Rα+ cells expressing low amounts of GFP were similar to those observed in control Linα4β7+IL-7Rα+Rorγthigh cells (Fig. 3D). There were milder and severe reductions in a proportion of α4β7+IL-7Rα+ LTi cells in Runx1P1N/P1N fetuses in anlagen for peripheral LNs (Supplemental Fig. 3), consistent with variation in the number of PPs and LNs. Importantly, low GFP expression in the α4β7+IL-7Rα+ cells was similarly observed in peripheral LN anlagen from the Cbfβ2m/2m fetuses (Supplemental Fig. 3). These results suggest that Cbfβ2 is likely to influence Rorγt gene expression rather than inhibit the differentiation of RorγtmidCD4lo/− cells into RorγthighCD4+ cells. However, GFP level in Lin lamina propria cells prepared from adult gut was unaffected by loss of Cbfβ2, although the proportion of these cells was severely reduced (Fig. 3E; M. Tachibana, unpublished observations). Thus, the effect of Cbfβ2 deficiency on Rorγt gene expression during LTi cell differentiation was apparent only in fetal anlagen, but not in adult tissue.

Normal Rorγt expression levels in Linα4β7+IL-7Rα+ LTi cells at anlagen in the absence of the P1-Runx1 variant suggest that some mechanism other than Rorγt regulation is involved in reducing the number of LTi cells in the Runx1P1N/P1N fetuses. Therefore, we examined the early differentiation of LTi precursors in fetal liver. A combination analysis of Rorγt and IL-7Rα expression detected two subsets in the Linα4β7+IL-7Rα+ population in the 13.5 dpc fetal liver. Based on the expression levels of IL-7Rα, the Lin α4β7+IL-7Rα+ subset could be subdivided into IL-7Rαmid and IL-7Rαhigh subset (Fig. 4A). Importantly, the emergence of α4β7+IL-7Rαhigh subset was first detected at the transition from 12.5–13.5 dpc embryonic day and was accompanied by the initiation of Rorγt expression (Fig. 4C). Thus, expression of Rorγt was observed only in the α4β7+IL-7Rαhigh subset. Consistent with expression profile of Rorγt, the α4β7+IL-7Rαhigh subset was absent in both Rorγt-deficient Rorγtgfp/gfp and the Id2−/− fetuses, whereas the α4β7+IL-7Rαmid subset was present (Fig. 4A, 4B). These results not only suggest a distinct characteristic between α4β7+IL-7Rαmid and α4β7+IL-7Rαhigh cells, but also identify the developmental stage at which the Rorγt and Id2 factors function to regulate LTi cell differentiation in fetal liver.

FIGURE 4.

Role of Runx1/Cbfβ2 complexes in differentiation of early LTi precursors in fetal liver. A, Expression profiles of α4β7 and IL-7Rα in Lin fetal liver cells from 13.5 dpc fetuses with the indicated genotypes. The numbers shown indicate the percentage of cells in the indicated region. B, Statistical summary of the flow cytometric analyses of multiple mice shown in A. The numbers in each panel (1–5) represent the control, Runx1P1N/P1N, Cbfβ2m/2m, Rorγtgfp/gfp, and Id2−/− genotypes, respectively. C, Expression profiles of GFP from the Rorγtgfp allele and IL-7Rα in Lin fetal liver cells from 12.5 and 13.5 dpc Rorγt+/+ and Rorγt+/gfp fetuses. D, Expression of B220 and CD19 in fetal liver cells from Runx1+/P1N and Runx1P1N/P1N 15.5 dpc embryo. Differentiation of B-lymphoid lineage cells was not affected by loss of P1-Runx1. E, Expression of c-Kit and Sca-1 in Lin fetal liver cells and absolute numbers of these Linc-Kit+Sca-1+ cells. These results indicate that the generation of multipotent hematopoietic progenitors is unaffected by loss of Cbfβ2 variant. Data are representative of two independent experiments. **p < 0.01.

FIGURE 4.

Role of Runx1/Cbfβ2 complexes in differentiation of early LTi precursors in fetal liver. A, Expression profiles of α4β7 and IL-7Rα in Lin fetal liver cells from 13.5 dpc fetuses with the indicated genotypes. The numbers shown indicate the percentage of cells in the indicated region. B, Statistical summary of the flow cytometric analyses of multiple mice shown in A. The numbers in each panel (1–5) represent the control, Runx1P1N/P1N, Cbfβ2m/2m, Rorγtgfp/gfp, and Id2−/− genotypes, respectively. C, Expression profiles of GFP from the Rorγtgfp allele and IL-7Rα in Lin fetal liver cells from 12.5 and 13.5 dpc Rorγt+/+ and Rorγt+/gfp fetuses. D, Expression of B220 and CD19 in fetal liver cells from Runx1+/P1N and Runx1P1N/P1N 15.5 dpc embryo. Differentiation of B-lymphoid lineage cells was not affected by loss of P1-Runx1. E, Expression of c-Kit and Sca-1 in Lin fetal liver cells and absolute numbers of these Linc-Kit+Sca-1+ cells. These results indicate that the generation of multipotent hematopoietic progenitors is unaffected by loss of Cbfβ2 variant. Data are representative of two independent experiments. **p < 0.01.

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In both Runx1P1N/P1N and Cbfβ2m/2m fetuses, the percentage of α4β7+IL-7Rαhigh subset was also severely reduced. However, in contrast to Rorγtgfp/gfp and Id2−/− fetuses, the percentage of the α4β7+IL-7Rαmid subset was also significantly reduced in the Runx1P1N/P1N and Cbfβ2m/2m fetuses (Fig. 4A, 4B), demonstrating that development of α4β7+IL-7Rαmid cells depends on the function of Runx1/Cbfβ2 complexes, but not on Rorγt or Id2.

In the Runx1P1N/P1N fetuses, we also observed a severe reduction in the α4β7IL-7Rα+ subset, which may contain multipotent hematopoietic progenitors. Given that Runx1/Cbfβ complexes are essential for generation of hematopoietic stem cells (22, 29), it is important to determine whether the reduction of the α4β7+IL-7Rαmid population in the Runx1P1N/P1N and Cbfβ2m/2m fetuses is caused by a reduced number of hematopoietic progenitors, as observed in Ikaros/ mice (31), or by the inhibition of progenitor differentiation into α4β7+IL-7Rαmid LTi precursors. Because differentiation potency toward both α4β7+IL-7Rα+ cells and B-lymphocyte lineage was detected in the Lin α4β7IL-7Rα+ fetal liver population in cell culture (13), we examined whether early B cell differentiation is impaired in the Runx1P1N/P1N fetuses. The development of B-lymphoid lineage cells in the 15.5 dpc fetal liver was detected as CD19+B220+ cells in both Runx1P1N/P1N and control fetuses (Fig. 4D). Furthermore, in the Cbfβ2m/2m fetuses, multipotent hematopoietic progenitors, defined as Linc-Kit+Sca-1+ cells, were increased rather than decreased in 13.5 dpc fetal liver (Fig. 4E), followed by an increased percentage of the α4β7IL-7Rα+ subset. These results indicate that development of multipotent progenitors was unaffected by Cbfβ2 deficiency and support the inference that Runx1/Cbfβ2 complexes are involved in the early specification of cells in the α4β7IL-7Rα+ population to the LTi lineage to force the differentiation of α4β7+IL-7Rαmid cells.

Based on the lack of both NK cells and LTi cells in the Id2−/− mice, it was assumed that Id2 is necessary for the development of the common precursors for both NK and LTi lineages (15, 32). Because the impaired differentiation of LTi precursor cells by Cbfβ2 deficiency was already observed prior to the developmental stage at which Id2 would function, we examined whether the differentiation of NK cells is affected in the Cbfβ2m/2m mice. NK cells, defined by their surface marker phenotype, were detected in the spleens and bone marrow of Cbfβ2m/2m mice and control mice (Fig. 5A), although the expression of some NK markers such as CD11b was lower in the Cbfβ2m/2m mice (Fig. 5B), as reported in mice expressing the dominant-negative form of Runx protein (33). Therefore, Cbfβ2 deficiency specifically inhibits the development of LTi cells but not NK cells.

FIGURE 5.

Presence of NK cells with downregulated NK markers in Cbfβ2m/2m mice. A, The proportions of NK cells, defined by the NK1.1+CD3ε surface phenotype, in the bone marrow (B.M.), spleen (Spl.), and liver (Liv.) were similar between littermate controls and Cbfβ2m/2m mice. B, Expression profiles of CD11b and DX5 in NK1.1+ cells showing the downregulation of CD11b with the loss of Cbfβ2 variant.

FIGURE 5.

Presence of NK cells with downregulated NK markers in Cbfβ2m/2m mice. A, The proportions of NK cells, defined by the NK1.1+CD3ε surface phenotype, in the bone marrow (B.M.), spleen (Spl.), and liver (Liv.) were similar between littermate controls and Cbfβ2m/2m mice. B, Expression profiles of CD11b and DX5 in NK1.1+ cells showing the downregulation of CD11b with the loss of Cbfβ2 variant.

Close modal

Hematopoietic LTi cells play an essential role in the development of second lymphoid tissue during embryogenesis. Albeit its unique function, an early differentiation pathway of LTi cells is not well understood. Results in this study revealed a novel function of Runx1/Cbfβ2 transcriptional factor complexes in lymphoid organogenesis in part via regulating LTi cell differentiation. There are two possible explanations why loss of P1-Runx1 and Cbfβ2 variant results in an impaired LTi development: reduced dosage of Runx1/Cbfβ expression or loss of variant-specific function. Although expression of both Cbfβ1 and Cbfβ2 transcripts are detected in many tissues at an almost similar ratio (25), expression of P1-Runx1 or P2-Runx1 was shown to be dominant in T-lineage cells or B-lineage and NK cells, respectively (24). Because we failed to examine expression of each Runx1 or Cbfβ variant protein due to a limited number of LTi cells in fetus tissues, dissection of these two possibilities waits for the future study. In both Runx1P1N/P1N and Cbfβ2m/2m mice, the formation of PPs and LNs was impaired, but was not completely abolished. This observation might suggest that Runx1/Cbfβ complexes are important, but not absolutely essential, for LTi cell development. However, because function of Runx1/Cbfβ complexes was only attenuated in the Runx1P1N/P1N and Cbfβ2m/2m mice, there remains a possibility that loss of entire Runx1/Cbfβ complex function completely inhibits LTi cell development, as was observed by the absence of the Rorγt or Id2 factor (15, 18).

Although the Rorγt and Id2 are known to be essential for the differentiation of LTi cells, the developmental stage at which these factors regulate LTi cell development is not well characterized. Our results show that Rorγt expression during LTi cell development is first detected in fetal liver cells of 13.5 dpc fetuses. Importantly, initiation of Rorγt expression correlates well with upregulation of the IL-7Rα expression. Thus, there are two cell subsets, Rorγtα4β7+IL-7Rαmid and Rorγt+α4β7+IL-7Rαhigh cells, in the Lin fetal liver population of 13.5 dpc fetuses. Together with the loss of the α4β7+IL-7Rαhigh subset in the Rorγtgfp/gfp and Id2−/− fetuses, Rorγt and Id2 factors potentially function at the same developmental stage to orchestrate differentiation of the α4β7+IL-7Rαmid cells into the α4β7+IL-7Rαhigh cells. Further studies are needed to confirm this developmental pathway and to understand how Rorγt and Id2 factors regulate development of the α4β7+IL-7Rαhigh subset. For instance, it is still unclear how upregulation of IL-7Rα is regulated at this transition. It is possible that Rorγt and Id2 factors are involved in regulating expression of the Il-7Rα gene. Alternatively, given that Runx1 is needed for efficient IL-7Rα expression during thymocyte differentiation (28), Runx1 may also play a role in the upregulation of IL-7Rα during LTi cell differentiation. However, the level of IL-7Rα expression on residual LTi cells is not impaired in the Runx1P1N/P1N and Cbfβ2m/2m mice, suggesting that remaining P2-Runx1 and Cbfβ1 variant could be sufficient for regulation of IL-7Rα in these mice, respectively.

In contrast, our results show that Runx/Cbfβ complexes are potentially involved in regulating expression of the Rorγt gene. We observed that the loss of Cbfβ2 variant results in a low level of GFP expression from the Rorγtgfp allele in residual α4β7+IL-7Rα+ LTi cells at lymphoid tissue anlagen. By using a surface CD4 expression as a marker to trace the differentiation pathway of LTi cells at anlagen, we showed the presence of two cell subsets, RorγtmidCD4low/− and RorγthighCD4+, in the control fetuses. Importantly, the expression level of CD4 in cells expressing a low level of GFP expression due to the Cbfβ2 deficiency was similar to that in control RorγthighCD4+ cells. This observation suggests that RorγtlowCD4+ cells in the Cbfβ2m/2m fetuses would correspond to the RorγthighCD4+ cells in control mice. Alternatively, given that Runx/Cbfβ complexes are essential for Cd4 gene repression via regulating the silencer activity in the Cd4 gene during T cell development (27, 34), if premature activation of the Cd4 gene occurs by the loss of Cbfβ2, RorγtmidCD4lo/− cells could be identified as RorγtmidCD4+ cells in the Cbfβ2m/2m mice. However, because there was no CD4 derepression in CD8+ T cells in the Cbfβ2m/2m mice (Y. Naoe and I. Taniuchi, unpublished observations), expression of the Cbfβ1 variant is sufficient for Cd4 gene repression at least in T lymphocytes, potentially excluding this possibility. We therefore conclude that Runx/Cbfβ2 complexes are needed for upregulation of the Rorγt gene expression in LTi lineage cells at anlagen. Although we failed to directly examine Rorγt expression in Linα4β7+IL-7Rαmid fetal liver cells due to a near absence of those cells in 13.5 dpc Cbfβ2m/2m fetuses, it is possible that Runx/Cbfβ2 complexes are also involved in initiation of the Rorγt expression in Linα4β7+IL-7Rαmid LTi precursors. The partial reduction of Rorγt expression suggests that Runx/Cbfβ2 complexes play a role in enhancing the Rorγt expression, as was observed in enhancing IFN-γ production in Th1 helper T cells by Runx3 protein (35). Our results indicated that level of Rorγt expression is unaffected by loss of the Cbfβ2 in adult Lin lamina propria cells. It is possible that only LTi cells that have successfully upregulated Rorγt expression in the absence of Cbfβ2 during embryogenesis can survive and accumulate as RorγthighCD4 cells in the adult lamina propria. Interestingly, Runx1 has been shown to be necessary for efficient Rorγt induction during the in vitro differentiation of Th17 cells (36). However, in our hands, the induction of Rorγt expression in both double-positive thymocytes and in vitro-differentiated Th17 cells was virtually unaffected by Cbfβ2 deficiency (I. Taniuchi, unpublished observations). Therefore, the mechanism regulating the Rorγt expression could differ between the LTi lineage cells and T lymphoid lineage cells. Further studies are necessary to unravel molecular mechanism regulating the developmental transition from Rorγtα4β7+IL-7Rαmid cells to Rorγt+#x03B1;4β7+IL-7Rαhigh cells.

Because the developments of both NK cells and LTi cells are defective in the Id2/ mice, Id2 was first supposed to regulate development of the common precursors for both NK and LTi lineages (15, 32). However, a recent study has shown that Id2-deficient mice retain a normal number of NK cell progenitors in adult bone marrow, suggesting that Id2 plays a role in the maturation of NK cells (37). Our results demonstrated that loss of Runx1/Cbfβ2 function impairs development of LTi cells before the developmental stage at which Id2 functions. However, we observed that development of NK cells is not significantly affected by loss of Cbfβ2. This result challenges the supposition that there exist common NK and LTi precursors for which development depends upon Id2. However, our results do not formally exclude the possibility that attenuation of Runx1/Cbfβ2 complex function results in a skewed differentiation of a common precursor toward NK lineage, as is observed in the cell-fate conversion of MHC class I-selected thymocytes toward helper lineage in part via dysregulated ThPOK transcription factor expression by loss of Runx/Cbfβ complex function (20). However, considering that an essential requirement of Cbfβ for NK cells development was reported by using hypomorphic Cbfβ allele (38), skewed differentiation toward NK lineage by the loss of Cbfb2 variant is unlikely. Nevertheless, because Runx/Cbfβ complexes function before Id2 during the differentiation of LTi precursors, Id2 is likely to differentially regulate the development of NK and LTi cells rather than play an essential role in the generation of common precursors for NK and LTi lineage.

In addition to the PPs, other types of mucosa-associated secondary lymphoid tissues, such as nasopharynx-associated lymphoid tissue and tear duct-associated lymphoid tissue, are identified. Interestingly, nasopharynx-associated lymphoid tissue organogenesis depends on Id2, but not Rorγt, expression (9, 39), whereas tear duct-associated lymphoid tissue formation is independent of both factors (40). Because results in this study revealed that Runx1/Cbfβ2 complexes act earlier than Id2 and Rorγt during differentiation of the LTi lineage cells, it is interesting to examine in the future study whether formation of another mucosa-associated secondary lymphoid tissues is impaired by loss of Runx/Cbfβ complexes function.

Collectively, our results demonstrate novel and essential roles for the Runx transcription factor complexes in regulating LTi cell development and provide insights into transcriptional regulation during the early specification stage toward the LTi-lineage (Supplemental Fig. 5). Further studies that seek for a deeper understanding of Runx-mediated specification to the LTi lineage would be beneficial for engineering artificial immune tissue.

We thank Dr. Dan R. Littman (New York University Medical Center) and Dr. Yoshifumi Yokota (University of Fukui) for providing the Rorγtgfp mice and Id2−/− mice, respectively. We also thank H. Wada for technical assistance in staining the NK cells and C. Miyamoto for mice genotyping.

This work was supported by grants from the Mitsubishi Foundation, the Uehara Memorial Foundation, and the Ministry of Education, Culture, Sports and Technology of Japan.

The online version of this article contains supplemental material.

Abbreviations used in this article:

α4β7

α4β7 integrin

dpc

days postconception

IL-7Rα

IL-7R α-chain

Lin

lineage marker-negative

LN

lymph node

LTi

lymphoid tissue inducer

LTo

lymphoid tissue organizer

P1

distal promoter

P2

proximal promoter

P1-Runx1

distal promoter-dervied Runx1 variant

P2-Runx1

proximal promoter-derived Runx1 variant

PP

Peyer’s patch

Rorγt

retinoic acid-related orphan receptor γt.

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The authors have no financial conflicts of interest.