Research ArticleImmunology

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

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Science Signaling  06 Oct 2015:
Vol. 8, Issue 397, pp. ra100
DOI: 10.1126/scisignal.aab2425

Sending thymocytes into action

MST1, the mammalian homolog of Hippo, plays a role in apoptosis and cellular proliferation by activating the kinase LATS, which inhibits the transcriptional coactivator YAP; however, MST1 also functions independently of LATS and YAP in T cell adhesion and migration. Tang et al. generated mice with a T cell–specific deficiency in both isoforms of the LATS-related kinase NDR. These mice had reduced numbers of naïve T cells in the periphery because mature thymocytes were trapped in the thymus. Chemoattractants stimulated actin polymerization and the migration of thymocytes in an MST1- and NDR-dependent manner, suggesting that the NDRs act downstream of MST1 to mediate thymocyte egress.

Abstract

The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell–specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.

INTRODUCTION

T lymphocytes in the circulation and in secondary lymphoid organs (SLOs) constitute one of the essential populations of cells that orchestrate and initiate effective adaptive immune responses. Maintenance of the T cell pool is dependent on a continuous supply of naïve T cells through the egress of mature thymocytes from the thymus (1, 2). Derived from bone marrow progenitor cells, thymocytes undergo a well-characterized developmental progression within the thymus to generate mature CD4 and CD8 single-positive (SP) thymocytes, which emigrate mainly through veins located at the corticomedullary junction and then traffic to SLOs (1, 3–5). Mature thymocyte emigration and lymphocyte migration in SLOs are processes that are guided collaboratively by a set of adhesion molecules and various signaling networks composed of sphingosine 1-phosphate (S1P) and chemokines, among which S1P and S1P receptor 1 (S1P1) form a key signaling module (4, 6–11).

MST1 (mammalian sterile 20–like kinase 1; also known as STK4), a ubiquitously expressed serine and threonine protein kinase, is the mammalian homolog of the Drosophila kinase Hippo (12, 13). In the current view of Hippo signaling, MST1 plays a critical role in apoptosis and proliferation through signaling by the kinase LATS (large tumor suppressor) and the transcriptional coactivator YAP (Yes-associated protein) (1215). Findings from studies of mouse models have established the kinase MST1 as a key mediator of T cell trafficking, adhesion, and survival (1620). Consistent with data from these studies, mutations in MST1 cause primary human immunodeficiencies (21, 22). The classical LATS-YAP signaling pathway downstream of MST1 does not seem to be critical in T cells because the phosphorylation status of LATS and YAP in T cells stimulated with S1P and chemokines is unaffected by the loss of MST1 and MST2 (MST1/2) (17, 18). Whereas dedicator of cytokinesis 8 (DOCK8)–MOB1–MST1 (18) and Rab13-MST1 (23) are two functional complexes that are thought to mediate MST signaling in lymphoid cells, the limited phenotypic overlap between mice deficient in DOCK8 or MST1/2 indicates that DOCK8 is only one of several contributing factors and that other downstream components of MST signaling in T cells are still largely unknown (2426).

The kinase LATS belongs to the nuclear Dbf2-related (NDR) family of kinases, which consists of another closely related subgroup of kinases, namely, NDR1 and NDR2. In addition to being similar to LATS in terms of primary structure, NDRs share with LATS the same upstream kinase MST in tissue culture systems (2729); however, the physiological importance of MST1 signaling upstream of NDRs, especially in immune cells, has not been addressed. The spontaneous development of a T cell lymphoma in aged NDR1-deficient mice supports a potential role for the NDRs in T cell homeostasis (30). However, no obvious T cell trafficking or proliferative deficiencies have been observed in these mice, which is most probably because of functional compensation by murine NDR2 (30). Thus, it is tempting to speculate that NDRs downstream of MST1 might play crucial roles in T cells.

Here, we generated and characterized an lck promoter–driven, cre recombinase–mediated, T cell–specific Ndr1/2 conditional double knockout (Ndr DKO) mouse model. Compared with control mice, the Ndr DKO mice exhibited a ~60% reduction in the number of their peripheral naïve T cells, and they displayed a phenotype consistent with impaired thymic emigration. We also found that the NDRs were activated in response to chemokines and S1P, as well as signaling downstream of MST1 during thymocyte emigration and the migration of naïve T cells. Collectively, these findings establish the kinases NDR1 and NDR2 as critical factors downstream of the kinase MST1 that mediate thymocyte egress and T cell migration.

RESULTS

T cell–specific deletion of the NDRs results in peripheral T cell lymphopenia

NDR1 is enriched in lymphoid organs, whereas NDR2 abundance is greatest in the small intestine and colon (30), and NDR1 and NDR2 functionally compensate for each in different tissues (30, 31). To study the roles of the NDRs in T cell development and function and to avoid any potential compensation between them, we generated T cell–specific Ndr1 and Ndr2 DKO mice by crossing Ndr1−/−Ndr2f/f mice with mice expressing cre recombinase driven by the lck proximal promoter. The deletion of both NDRs in Ndr1−/−Ndr2f/f/lck-cre mice (hereafter referred as Ndr DKO mice) was validated by Western blotting analysis of thymus extracts (Fig. 1A). Ndr DKO mice were fertile and born at a Mendelian ratio, and no obvious morphological or behavioral abnormalities were observed in young adult Ndr DKO mice (8 to 16 weeks of age).

Fig. 1 T cell–specific deletion of Ndr1/2 results in peripheral T cell lymphopenia.

(A) Lysates from the thymi of the indicated wild-type (WT) and KO mice were analyzed by Western blotting with antibodies against the indicated proteins. Actin was used as a loading control. Data are representative of three experiments. (B and C) T cells from the spleens (B) and lymph nodes (C) of WT and Ndr DKO mice were analyzed by flow cytometry to determine the percentages of CD4+ and CD8+ cells, which are indicated. Dot plots are representative of five experiments. (D and E) Quantification of the percentages (D) and total numbers (E) of CD4+ and CD8+ T cells in the spleens of WT, Ndr1 KO, Ndr2 KO, and Ndr DKO mice, as indicated. Data are means ± SD of five mice of each genotype from five experiments. (F and G) Quantification of the percentages (F) and total numbers (G) of CD4+ and CD8+ T cells in the lymph nodes (LNs) of the indicated mice. Data are means ± SD of three mice of each genotype, except for Ndr DKO mice (n = 4), from three experiments. (H) Quantification of the numbers of naïve and memory CD4+ and CD8+ T cells in the spleens of the indicated mice. Data are means ± SD of five mice of each genotype from five experiments. (I) Quantification of the numbers of naïve and memory CD4+ and CD8+ T cells in the lymph nodes of the indicated mice. Data are means ± SD of three mice of each genotype from three experiments. For (D) to (I): *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant.

We first compared the T cell composition in the SLOs of Ndr DKO mice with that of control wild-type mice and found that the percentages of CD4+ and CD8+ T cells in the SLOs of the Ndr DKO mice were decreased (Fig. 1, B to D). Quantification of the absolute numbers of CD4+ and CD8+ cells in the spleen revealed that Ndr DKO mice had statistically significant reductions of about 30 and 20%, respectively, compared to those of wild-type mice (Fig. 1E). We also observed substantial reductions in the total numbers of CD4+ and CD8+ T cells in the lymph nodes of Ndr DKO mice (Fig. 1, C, F, and G).

To further distinguish the contributions of NDR1 and NDR2 in T cells, we analyzed Ndr1 or Ndr2 single KO mice and compared them with wild-type mice. We did not observe any statistically significant difference in the ratios or total numbers of peripheral CD4+ and CD8+ T cells between wild-type mice and either Ndr1-deficient or Ndr2-deficient mice (Fig. 1, D to G). Moreover, no significant differences were observed between wild-type mice and mice with one single allele of either Ndr1 (Ndr1−/+Ndr2f/f/lck-cre) or Ndr2 (Ndr1−/−Ndr2f/+/lck-cre) (fig. S1A), indicating a fully functional role for a single allele of Ndr1 or Ndr2 in vivo. Our results suggested that the effect on T cell number that we observed was NDR-specific, because mice expressing only cre recombinase (wild-type/lck-cre mice) did not show any phenotype (fig. S1A). This result confirmed the functional compensation between NDR1 and NDR2 and further indicated that single deletion of Ndr1 or cre-mediated deletion of Ndr2 had no substantial effect on T cell homeostasis. Thus, in some of our subsequent experiments, we used Ndr2 KO mice as an additional control. Our observation of the decreased total numbers of CD4+ and CD8+ T cells in young Ndr DKO mice, together with the previously reported development of T cell lymphoma in aged Ndr1 KO mice (30), suggests that the NDRs play a critical role in maintaining T cell homeostasis and that the loss of NDRs in T cells results in lymphopenia.

Loss of NDRs results in decreased numbers of naïve T cells

Loss of peripheral T cells usually arises from three major possibilities: increased ongoing apoptosis, impaired proliferation, or decreased thymic export. We first analyzed the ongoing apoptosis of freshly isolated lymphocytes from the spleen or lymph nodes. Staining of CD4+ lymphocytes with annexin V followed by flow cytometric analysis did not reveal any substantial difference in apoptosis between wild-type, Ndr2 KO, or Ndr DKO mice (fig. S1B). These findings suggest that an increase in apoptosis is unlikely to explain the lymphopenia observed in Ndr DKO mice.

NDRs have been depicted as critical cell cycle regulators by phosphorylating p21 and stabilizing c-myc (32, 33). Thus, we next asked whether Ndr DKO T cells displayed a proliferative defect. To this end, we performed in vivo 5-bromo-2′-deoxyuridine (BrdU) incorporation assays to analyze the proliferation of T cells in lymph nodes. We observed an increase in BrdU-containing (BrdU+) CD4+ T cells from 0.75% in wild-type control mice to 1.6% in Ndr DKO mice (fig. S1, C and D). Peripheral CD4+ and CD8+ T cells can be further divided into naïve (CD62highCD44low) and memory (CD44high) populations. To further analyze whether there was an intrinsic difference between the T cells from control mice and Ndr DKO mice, we studied the BrdU+ cell populations in naïve and memory T cells. However, we did not observe any statistically significant difference in the numbers of these populations between wild-type and Ndr DKO mice (fig. S1, C and D), which suggests that the increase in the total number of CD4+ T cells did not result from an intrinsic proliferative advantage, but instead reflected differences in the numbers of the naïve and memory cell populations between the different mice. We further quantified the naïve and memory T cells in our experimental groups and found that there was an increase in the percentage of CD44high memory T cells in Ndr DKO mice compared to that in wild-type mice (fig. S1, E and F). Analysis of the total numbers of cells in each population revealed an about 60% decrease in the numbers of naïve CD4+ and CD8+ splenocytes in Ndr DKO mice compared to those in wild-type mice (Fig. 1H). We observed a similar pattern of reduced numbers of naïve T cells and a homeostatic increase in the numbers of memory T cells in lymph nodes from Ndr DKO mice (Fig. 1I). The differentiation of T cells from naïve cells to memory-like cells is consistent with a homeostatic mechanism resulting from lymphopenia-induced proliferation (34, 35). Collectively, these results suggest that NDRs play a critical role in maintaining the homeostasis of peripheral naïve T cells.

Mature SP thymocytes accumulate in Ndr DKO mice

The results from our analysis of T cell apoptosis and proliferation (fig. S1, B to D) cannot explain the loss of peripheral T cells in DKO mice (Fig. 1). Because the number of cells released from the thymus (“thymic output”) also affects the homeostasis of the peripheral T cell pool, we next investigated the thymic composition in DKO mice. Although we did not observe any difference in thymic cellularity among any of the different mouse strains (Fig. 2A), we found that Ndr DKO mice had an increased ratio of CD4SP to CD8SP cells compared to those of wild-type and Ndr2 KO control mice (Fig. 2, B and C). Further quantification of the numbers of CD4+ (CD3+CD4+) and CD8+ (CD3+CD8+) SP thymocytes in DKO mice indicated that they were increased by about 2- and 1.2-fold, respectively, compared to those in wild-type mice (Fig. 2D). There were no differences in the numbers of double-negative (DN; CD3CD4CD8) or double-positive (DP; CD4+CD8+) thymocytes among the different mouse strains (Fig. 2D). These observations suggest that there is an increase in the numbers of CD4SP and CD8SP thymocytes in Ndr DKO mice.

Fig. 2 Ndr DKO mice have increased numbers of SP thymocytes.

(A) Quantification of the total numbers of thymocytes (thymic cellularity) in the thymi of the indicated mice. Data are means ± SD of four mice of each genotype from four experiments. (B) Thymocytes from WT and Ndr DKO mice were subjected to flow cytometric analysis of the relative percentages of CD4SP and CD8SP cells, as indicated. Dot plots are representative of four experiments. (C) Quantification of the percentages of CD4SP and CD8SP thymocytes in the thymi of the indicated mice. Data are means ± SD of four mice of each genotype from four experiments. (D) Quantification of the total numbers of DP (CD4+CD8+), DN (CD3CD4CD8), CD4SP (CD3+CD4+CD8), and CD8SP (CD3+CD4CD8+) cells in the indicated mice. Data are means ± SD of four mice of each genotype from four experiments. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

To further characterize the increased numbers of SP thymocytes in Ndr DKO mice, we examined the developmental profile of Ndr DKO SP thymocytes to determine which population of SP thymocytes was affected by loss of the NDRs. During the maturation process, SP thymocytes increase their cell surface abundances of CD62L and S1P1, whereas they decrease the amounts of CD69 and CD24. We used flow cytometric analysis with antibodies specific for CD24, CD62L, and CD69 to identify mature SP thymocytes (CD24lowCD62LhighCD69low), and we observed a statistically significant increase in the percentages of mature CD4SP and CD8SP thymocytes in Ndr DKO mice compared to those in wild-type mice (Fig. 3A and fig. S2A). Furthermore, we detected a similar pattern of increased cell surface abundance of S1P1 on mature SP thymocytes from wild-type, Ndr2 KO, and Ndr DKO mice (Fig. 3B). Further quantification of cell numbers revealed that there was a statistically significant increase in the numbers of mature CD4SP and CD8SP thymocytes in the Ndr DKO mice (Fig. 3, C and D). We did not detect any differences in the numbers of immature CD4SP and CD8SP (CD24highCD62LlowCD69highS1P1low) thymocytes among the different mouse strains (Fig. 3D). These results demonstrated the accumulation of mature SP thymocytes in mice deficient in NDR1 and NDR2.

Fig. 3 Ndr DKO mice have increased numbers of mature SP thymocytes.

(A) CD4SP (top) and CD8SP (bottom) thymocytes from WT and Ndr DKO mice were gated by flow cytometric analysis into immature (CD62LlowCD69high) and mature (CD62LhighCD69low) populations (dot plots, left) and then were assessed for their cell surface abundance of S1P1 (histograms, right). (B) Analysis of S1P1 abundance on immature and mature CD4SP and CD8SP thymocytes from the indicated mice represented in (A). Histograms are representative of four experiments. (C) The numbers of mature CD4SP and CD8SP thymocytes are expressed as percentages of the total numbers of SP thymocytes in the indicated mice. Data are means ± SD of four mice of each genotype from four experiments. (D) Quantification of the total numbers of immature and mature CD4SP (left) and CD8SP (right) in the indicated mice. Data are means ± SD of four mice of each genotype from four experiments. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

Thymic egress is impaired in Ndr DKO mice

An increase in the size of the mature SP population might result from (i) a defect in egress from the thymus; (ii) increased proliferation of mature SP thymocytes; (iii) enhanced maturation transition from an earlier stage (that is, DP cells to immature SP cells and then to mature SP cells); (iv) enhanced recirculation of peripheral T cells back to the thymus; or (v) a failure in negative selection in the thymus. We did not observe any obvious difference between young Ndr DKO and wild-type mice (up to 16 weeks old), such as in body weight or spleen weight (fig. S2B), which suggested that negative selection and immune tolerance might be normal in young Ndr DKO mice. Thymus recirculating peripheral T cells are restricted to activated T cells and are phenotypically characterized as CD62LlowCD69low cells (36). We found that the percentages of CD62LlowCD69low cells (2 to 3%) within the SP thymocyte populations of wild-type mice and Ndr DKO mice were similar (Fig. 3A), which seemed to rule out the possibility of there being increased recirculation of peripheral T cells in the Ndr DKO mice.

To examine the individual contributions of the processes of emigration, proliferation, and transition to the accumulation of mature SP thymocytes in Ndr DKO mice, we performed in vivo BrdU pulse-chase assays to assess the dynamics of thymocyte development (Fig. 4 and fig. S2). BrdU is incorporated into proliferating cells, which in the thymus are mainly DN, early DP, and a small number of mature SP populations. One day after we injected wild-type mice with BrdU, we found that 4.5% of mature CD4SP cells were BrdU+, whereas only 1.5% of mature CD4SP cells in Ndr DKO mice were BrdU+; however, BrdU incorporation into other subsets of cells from wild-type and Ndr DKO mice was similar (Fig. 4, A and B, and fig. S2C). This result suggests that enhanced proliferation is unlikely a reason for the accumulation of mature SP thymocytes in DKO mice.

Fig. 4 Loss of NDR1/2 impairs the emigration of mature thymocytes from the thymus.

(A to D) WT and Ndr DKO mice were injected with BrdU as described in Materials and Methods. On the indicated days after injection, the incorporation of BrdU into immature (red, CD62LlowCD69highCD24highCD4+) and mature (blue, CD62LhighCD69lowCD24lowCD4+) CDSP thymocytes was assessed by flow cytometric analysis. (A) Representative flow cytometry histograms for cells analyzed on the indicated days after injection. (B) Quantitative analysis over time of the percentages of BrdU+ cells among the indicated populations. (C and D) Representative flow cytometry histograms (C) and quantitative analysis (D) of the incorporation of BrdU into naïve and memory CD4+ T cells in the peripheral lymph nodes at the indicated times after the mice were injected with BrdU. Data in (B) and (D) are means ± SD of three to four mice per experiment and are representative of four experiments. *P < 0.05, **P < 0.01.

We next investigated the dynamics of transitions in thymocyte development by analyzing on days 4 and 7 after injection of the mice with BrdU. On day 4 after injection, the positively selected DP thymocyte population had transitioned into immature SP thymocytes. We found that about 40% of immature cells in all mice were BrdU+ (Fig. 4, A and B), which suggests that the maturation transition from DP thymocytes to immature SP thymocytes was not affected by the loss of NDRs. On day 7 after injection, the immature SP thymocytes had developed into mature SP thymocytes, which would normally migrate to the periphery. We observed similar kinetics of decreases in the percentages of BrdU+ thymocytes within the immature SP thymocyte population of wild-type and Ndr DKO mice (Fig. 4B, left), which suggested that the maturation transition from immature SP thymocyte to mature SP thymocyte was indistinguishable in both mouse strains (Fig. 4, A and B). Collectively, these results suggest that maturation transition from days 4 to 7 is comparable between wild-type and Ndr DKO mice.

Because the average intrathymic life span of SP thymocytes is about 12 days (37), we further analyzed the mice 12 days after they were injected with BrdU to study the dynamics of the emigration of BrdU+ mature SP thymocytes from the thymus and their trafficking to the periphery. Whereas about 6% of mature CD4SP thymocytes in wild-type mice were BrdU+ on day 12, about 10% of these cells in Ndr DKO mice were BrdU+ (Fig. 4, A and B). Consistent with a delayed thymic exit, the percentage of BrdU+ naïve T cells in lymph nodes was substantially reduced in Ndr DKO mice (Fig. 4, C and D), which suggests that there was a reduction in the number of recent thymic emigrants in the periphery as a result of impaired emigration of mature thymocytes from the thymus, which was already notable on day 7 after injection with BrdU (Fig. 4, C and D). Together, the results from these in vivo BrdU incorporation experiments exclude enhanced proliferation or accelerated maturation transition as contributing factors to the increased accumulation of mature SP thymocytes in the Ndr DKO mice. Instead, our findings suggest that a thymic emigration defect in the Ndr DKO mice results in the accumulation of mature SP thymocytes in the thymus and a decrease in the number of peripheral naïve T cells.

Defects in thymocyte emigration result in the accumulation of SP thymocytes in the perivascular space of the thymus, which is characterized as the area around the blood vessel that is surrounded by mesenchymal cells that are stained with ER-TR7, an antibody that recognizes reticular fibroblasts and reticular fibers (38). To determine the spatial localization of mature thymocytes, we performed immunofluorescence microscopy analysis of thymic sections (fig. S3). Thymic mesenchymal cells, including perivascular cells and fibroblasts, were detected with an anti–ER-TR7 antibody, whereas thymic medullary epithelial cells were identified by their expression of keratin 5. Consistent with there being an impairment in thymocyte egress in the Ndr DKO mice, we found enlarged medullary regions with abundant CD3+ cells, which accumulated in the perivascular space between the epithelial cells and the ER-TR7+ perivascular cells (fig. S3). This distribution pattern mimics that observed during S1P1 deactivation in mice treated with S1P1 modulator FTY720, which blocks thymocyte egress (38), and suggests that the chemotactic emigration of Ndr DKO mature thymocytes in response to venous S1P was impaired. Together, our results suggest a critical role for NDRs in the emigration of mature thymocytes.

The impaired chemotactic response of T cells is associated with a defect in actin polarization and Rho guanosine triphosphatase activity

The emigration of mature thymocytes across the vascular endothelium is guided by a gradient in the concentration of venous S1P. We further analyzed the chemotactic migration of Ndr DKO thymocytes with Transwell assays. Whereas control thymocytes from wild-type and Ndr2 KO mice migrated similarly to both S1P and the chemokine CCL19, the response of Ndr DKO SP thymocytes to S1P was substantially impaired and the numbers of Ndr DKO SP thymocytes that migrated in response to CCL19 were decreased by ~50% compared to that of control cells (Fig. 5A). These results suggest that NDRs are critical to ensure normal chemoattractant-stimulated migration. Note that the reduced chemotactic migration of Ndr DKO thymocytes was accompanied by the failure of both S1P and CCL19 to stimulate polarization of F-actin (Fig. 5, B to D).

Fig. 5 Chemotactic migration and Rho-dependent actin polarization are impaired in Ndr DKO thymocytes.

(A) CD4SP or CD8SP thymocytes from the indicated mice were analyzed in Transwell migration assays for chemotaxis toward the indicated concentrations of S1P (left and middle) or CCL19 (200 ng/ml, right). Data are means ± SD of three experiments. (B) Thymocytes from WT or Ndr DKO mice were left unstimulated [phosphate-buffered saline (PBS)] or were stimulated for 2 min with 100 nM S1P or CCL19 (200 ng/ml) before being stained with phalloidin and analyzed by confocal microscopy to assess F-actin polarization. Images are representative of three experiments. Scale bar, 2 μm. (C and D) Quantification of the numbers of thymocytes (C) and sorted CD4SP thymocytes (D) with a polarized distribution of F-actin. Data were obtained by analyzing cells from five randomly selected fields of view. (E) WT and Ndr DKO thymocytes were unstimulated or were stimulated with S1P or CCL19 before being subjected to the pull-down of active RhoA, which was followed by Western blotting analysis with an antibody specific for total RhoA. Western blots are representative of three experiments. Right: Relative fold changes in the abundance of active Rho normalized to that of total RhoA were quantified by densitometric analysis with ImageJ software. Data are means ± SD of three experiments. *P < 0.05, **P < 0.01.

Changes in the actin cytoskeleton require the activation of members of the Rho family of guanosine triphosphatases (GTPases). For example, S1P and CCL19 stimulate the transient activation of RhoA (39), which initiates the actin polarization–mediated cell migration. We next asked whether there was an intrinsic defect in Rho activity upon loss of NDRs. Indeed, whereas there was an increase in the abundance of guanosine 5′-triphosphate (GTP)–bound (active) RhoA in wild-type thymocytes in response to CCL19 or S1P, the abundance of GTP-bound RhoA in Ndr DKO thymocytes was decreased under both unstimulated and stimulated conditions (Fig. 5E). Together, our findings suggest that the NDRs play a role in mediating the emigration of mature thymocytes and that loss of NDRs impairs the RhoA-stimulated polarization of the actin cytoskeleton, which results in decreased migration and the accumulation of mature SP thymocytes around the perivascular space and consequently a decrease in the number of naïve T cells in the periphery of Ndr DKO mice.

NDRs are required for T cell motility in vivo

To further validate the contribution of NDRs to T cell motility, we first analyzed the role of NDRs in the trafficking of naïve T cells to SLOs. To this end, naïve T cells were isolated from wild-type, Ndr2 KO, and Ndr DKO mice, fluorescently labeled, and adoptively transferred into wild-type recipient mice, and their distribution to the spleen, peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches was analyzed. This analysis revealed that the homing of Ndr DKO naïve T cells was not decreased compared to that of Ndr2 KO naïve T cells (fig. S4A). This finding suggests that the tethering, rolling, and firm adhesion of the naïve T cells were not adversely affected by the loss of NDR proteins. This notion was supported by the finding that the cell surface abundances of CCR7 (the receptor for CCL19) and the integrin lymphocyte function–associated antigen–1 (LFA-1) [the receptor for intercellular adhesion molecule–1 (ICAM-1)] were similar on Ndr DKO T cells and wild-type T cells (fig. S4B). We consistently observed the enhanced homing of Ndr DKO naïve T cells to Peyer’s patches (fig. S4A). CCR7 and the integrin α4β7 specifically contribute to the homing efficiency of naïve T cells to Peyer’s patches (6). We found that the cell surface abundance of CCR7 was similar for mature SP thymocytes from wild-type and Ndr DKO mice (fig. S4B); however, the abundance of α4β7 was greater on mature SP thymocytes from Ndr DKO mice (fig. S4B), which may explain their enhanced homing to Peyer’s patches.

Next, we investigated the dynamics of the interstitial migration of naïve T cells in the T cell area of the thymus by two-photon microscopy (Fig. 6). High endothelial venules (HEVs) were labeled to distinguish the T cell areas from the parenchyma. We found that loss of NDRs markedly decreased the dynamics of thymocyte migration within the parenchyma of the popliteal lymph node (Fig. 6A). Ndr DKO naïve T cells were mostly stationary and did not display the random guided walk exhibited by wild-type and Ndr2 KO T cells. Furthermore, the Ndr DKO naïve T cells did not exhibit the elongation and polarity changes displayed by the wild-type and Ndr2 KO cells (Fig. 6, A and B). Further quantification revealed that the mean speed of migration of wild-type cells through the parenchyma was 10.84 ± 3.63 μm/min, whereas for Ndr DKO cells it was 8.89 ± 4.02 μm/min (Fig. 6C). Similarly, the mean meandering index was substantially reduced upon loss of the NDRs, from 0.63 ± 0.24 for wild-type cells to 0.43 ± 0.30 for Ndr DKO cells (Fig. 6D). Accordingly, the motility coefficient of Ndr DKO naïve T cells was decreased by ~60% compared to those of wild-type and Ndr2 KO naïve T cells (Fig. 6, E and F). Together, these data suggest that in addition to playing a role in the emigration of mature thymocytes, the NDRs mediate the interstitial migration of naïve T cells in peripheral lymph nodes.

Fig. 6 NDR1 and NDR2 mediate T cell migration in vivo.

(A to F) CD44low-med T cells from WT, Ndr2 KO, or Ndr DKO mice were fluorescently labeled and adoptively transferred into C57BL/6 recipient mice for two-photon microscopic imaging of popliteal lymph nodes. (A) Sequence of two-photon microscopic images showing representative WT (white dashed lines), Ndr2 KO (red dashed lines), and Ndr DKO (arrowheads) cells. Scale bar, 20 μm. Time is given in minutes and seconds. (B) Examples of typical cell shapes observed in the two-photon microscopic image sequences. The blue arrow indicates the track and direction of cell migration. The tracks of WT and Ndr2 KO cells represent shapes acquired over a time span of 8 min and 20 s, whereas the tracks of Ndr DKO cells represent a time span of 30 min. Scale bar, 20 μm. (C) Track speeds of 10.84 ± 3.63, 11.58 ± 3.35, and 8.89 ± 4.02 μm/min were recorded for single-cell tracks of WT, Ndr2 KO, and Ndr DKO cells, respectively. Red bars indicate medians. (D) Meandering index values of 0.63 ± 0.24, 0.67 ± 0.24, and 0.43 ± 0.30 were recorded for WT, Ndr2 KO, and Ndr DKO cells, respectively. Red bars indicate medians. (E) Mean displacement of WT, Ndr2 KO, and Ndr DKO T cells as a function of the square root of time. (F) Motility coefficients of the WT, Ndr2 KO, and Ndr DKO T cell populations. Data in (C) to (E) are pooled from two-photon microscopy image sequences obtained from a total of four recipient mice from two independent experiments, each of which involved three WT mice, two Ndr2 KO mice, and three Ndr DKO mice. ***P < 0.001.

NDRs signal downstream of the kinase MST1 in T cells

Our earlier findings suggested that Ndr-deficient mice phenocopy MST-deficient mice in terms of impaired emigration of mature thymocytes and interstitial migration of lymphocytes in popliteal lymph nodes (16, 18, 19). Because MST1 signaling is activated by chemokines and S1P (18), we examined whether the NDRs were also activated in response to the same chemotactic factors. To this end, we treated thymocytes with S1P, the predominant driver of thymic emigration, and we analyzed the activation of NDRs by Western blotting analysis of phosphorylated NDR1/2 with an antibody specific for the hydrophobic motif phosphorylation site of NDRs, which is essential for the kinase activity of the NDRs (40, 41). We first found that the NDRs were phosphorylated in thymocytes in response to S1P (Fig. 7A and fig. S5A). Consistent with previous reports (17, 18), the phosphorylation of MST and MOB1 was stimulated by S1P (Fig. 7A and fig. S5A). Similar to S1P, both CCL19 and CCL21 (ligands for CCR7) stimulated the phosphorylation (and activation) of MST and NDR1/2 in thymocytes (Fig. 7A and fig. S5A).

Fig. 7 NDRs function downstream of MST.

(A) Thymocytes from WT mice were stimulated with 100 nM S1P for the indicated times (left) or were stimulated with the indicated agonists for 5 min (right) before being subjected to Western blotting analysis with antibodies specific for the phosphorylated forms of NDR1/2, MST1, and MOB1. Tubulin was used as a loading control. Western blots are representative of three experiments. (B) Single-cell suspensions of thymocytes from WT mice were prepared in RMPI 1640 medium supplemented with 2% fetal calf serum (FCS). After incubation with PBS, S1P, or CCL19 for 5 min, the thymocytes were lysed and subjected to immunoprecipitation with an anti-mNDR1 antibody or with IgG as a negative control. Immunoprecipitated samples and whole-cell lysates (Input) were then analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots are representative of three experiments. (C) Thymocytes from WT and MST1 KO mice were left unstimulated or were stimulated with CCL19 or S1P for 5 min. Whole-cell lysates were then prepared and subjected to Western blotting analysis of the indicated components of the Hippo signaling pathway. Western blots are representative of three experiments. (D) Proposed model for the role of NDRs in thymocyte emigration and lymphocyte motility. S1P and chemokines activate the MST-hMOB-NDR complex for efficient emigration of thymocytes from the thymus and interstitial migration of lymphocytes in lymph nodes. Loss of MST or NDRs results in impaired emigration and motility. Additionally, Rap1-RapL activates MST to initiate integrin activation and effective lymphocyte trafficking. Other upstream signals may activate NDRs during T cell migration.

Given that MST1 functions upstream of NDRs in transformed tissue culture cells (27, 29) and that it facilitates the interaction between MOB1 and NDR (27, 42), we speculated that NDR might function downstream of MST-MOB1 signaling in T cells. Coimmunoprecipitation experiments showed that both CCL19 and S1P stimulated the formation of complexes containing NDR1, MST1, and MOB1 in wild-type thymocytes (Fig. 7B and fig. S5, B and C). To define whether the NDRs indeed functioned downstream of MST1 in T cells, we investigated the chemokine- and S1P-dependent activation of NDRs in MST1-deficient thymocytes (Fig. 7C). Consistent with previous reports (17, 18), the LATS-YAP signaling axis was largely unaffected by stimulation with CCL19 or S1P or by the loss of MST1 (Fig. 7C). However, the CCL19- and S1P-stimulated phosphorylation of the NDRs was inhibited in Mst1 KO thymocytes, as was the phosphorylation of MOB1 (Fig. 7C). Together, these data suggest that NDRs may function downstream of MST1 signaling in T cells (Fig. 7D).

DISCUSSION

Here, we reported the roles of NDR1/2 in mature thymocyte emigration and T cell migration in vivo. A defect in thymocyte egress caused by the loss of NDR1/2 led to the accumulation of mature thymocytes in the perivascular space within the thymus, which partially mimics the phenotype caused by S1P1 blockade with FTY720. Consequently, the number of naïve T cells in the SLOs of Ndr DKO mice was reduced by ~60% compared to those of wild-type mice; however, the number of memory T cells was maintained because of homeostasis. We further defined the contribution of the NDRs to T cell motility in popliteal lymph nodes. Whereas T cells from wild-type mice were motile and displayed an elongated morphology and changes in polarity, Ndr DKO T cells were stationary and had a ~60% decrease in their motility coefficient. Moreover, we showed that NDRs were activated in response to chemokines and S1P and that they were required for optimal chemokine-induced Rho activation and actin polarization. Together, these findings are suggestive of critical roles for NDR1/2 downstream of the kinase MST1 in mediating efficient T cell motility.

Chemokine receptors and other heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) collaboratively guide lymphocytes to migrate into, within, and out of the thymus and SLOs. S1P and its receptor S1P1 play fundamental roles in the emigration of lymphocytes from the thymus and SLOs (9–11, 43). Despite the identification of several intracellular components of the S1P1 signaling pathway, those molecules downstream of the receptor that initiate cytoskeletal changes and migration are still largely unknown (7). The kinase MST1 was identified as one of the key intracellular molecules that mediate thymocyte emigration and lymphocyte trafficking in mice (16, 18, 19). Mutations in the gene encoding MST1 in humans cause a primary immunodeficiency that is characterized by the loss of naïve T cells (21, 22); however, the mechanism by which the loss of MST1 induces the depletion of peripheral naïve T cells is still largely unknown. Here, we demonstrated that the NDRs can function as physiological downstream targets of MST1 signaling.

NDRs are implicated as downstream targets of MST1 in cultured cells in vitro (2729); nevertheless, direct physiological evidence of their role is still lacking. Here, we identified a defect in thymocyte emigration and interstitial migration caused by Ndr deficiency that mimics the phenotypes of Mst1 KO mice (16, 18, 19), which suggests that NDRs are downstream of MST in vivo. This notion was further supported by our demonstration that NDRs form a ternary complex with MST1 and MOB1. Moreover, the dependence of NDR1/2 kinase activity on MST1 signaling further supports the role of the MST1-NDR pathway in T cells. Together, our data suggest that NDRs may be targets of MST1 in cells of the immune system.

The interstitial migration of lymphocytes in lymphoid organs, which is central to their ability to search for antigens and initiate adaptive immunity, is dependent on actin cytoskeletal rearrangements and cell polarization events (7, 8). MST proteins mediate lymphocyte interstitial migration by stimulating Rho GTPase activity (16, 18). Loss of MST1 results in the failure of chemokine- and S1P-induced actin polarization in T cells (1619). We further explored the potential contribution of NDRs to the interstitial migration of T cells and demonstrated the role for NDR1/2 downstream of MST1 in mediating Rho activation and migration.

One notable difference between Mst KO mice and Ndr DKO mice is in the process of T cell homing. MST1 mediates L-selectin–dependent tethering and rolling (16), clustering of LFA-1 (17), and ICAM-1–dependent adhesion and migration (16, 20), and thus stimulates lymphocyte homing downstream of Rap1-RapL/Nore1B (44). Our observation that Ndr DKO mice had no defect in the homing of naïve T cells suggests that the cell surface amounts of CD62L, LFA-1, CCR7, and CXCR4 and the availability of their ligands, which shapes lymphocyte homing, might not be affected by the loss of NDR1/2. Indeed, we found that the cell surface abundances of LFA-1 and CCR7 were comparable between Ndr DKO T cells and wild-type T cells. Furthermore, the cell surface abundance of the integrin α4β7, which determines the homing efficiency specifically to Peyer’s patches, was increased on Ndr DKO T cells, which correlated with the enhanced homing of these cells to Peyer’s patches. When activated by MST1, hMOB1 interacts with DOCK8 (18), an essential activator of CDC42 (45). It is possible that hMOB1 forms two pools in T cells, hMOB1-NDR and hMOB1-DOCK8, which control distinct functions. Rab13 is another MST1-interacting protein and an effector of MST1 during T cell trafficking and homing (23). All of these studies suggest that Rab13 (23), hMOB1-DOCK8 (18), or other uncharacterized factors may function in parallel to NDRs downstream of MST1 signaling in different aspects of T cell function. Therefore, future research into dissecting MST1-NDR signaling in the context of the functions of Rab13 and DOCK8 is warranted.

Our Ndr DKO mice displayed a different level of lymphopenia than that of the Mst KO mice, which could be a result of the different effects of MST and NDR on T cell apoptosis (17, 18). Whereas increased apoptosis was described in one MST-deficient mouse, we only observed a slight, but not statistically significant, increase in apoptosis in the Ndr DKO mice. This difference suggests that other molecules, such as FOXO (forkhead box O), might be direct targets of MST1 during T cell apoptosis (46). Another possibility is that the NDRs reduce only the efficiency of thymocyte migration. Alternatively, the effect of loss of the NDRs could be compensated for by the activity of other components, such as Rab13 (23).

MST1 prevents the development of lymphoma by promoting faithful chromosome segregation (47). In this context, it is noteworthy that NDRs have also been implicated in hematological malignancies (30, 48). Additionally, NDRs play essential roles in chromosome alignment and mitotic progression (28, 49, 50). Thus, future work into examining the contribution of NDRs to lymphomagenesis induced by an MST1 deficiency is warranted. Those findings will shed light on the coordinated regulation of lymphocyte development, trafficking, and lymphomagenesis by the MST-NDR signaling pathway, which would further help us to appreciate how homeostasis of the immune system is maintained and how the development and trafficking of lymphocytes is connected to pathological primary immunodeficiencies and hematological tumorigenesis.

MATERIALS AND METHODS

Mice

Ndr DKO mice were generated by crossing Ndr1−/−Ndr2f/f mice with mice expressing cre recombinase driven by the lck proximal promoter [Jackson Laboratory, B6.Cg-Tg (Lck-cre; 548Jxm/J stock #003802)]. All of the mice used for experiments were backcrossed to C57BL/6J mice for at least six generations. The C57BL/6J mice used for adoptive transfer experiments were purchased from Charles River. Young adult mice (8 to 12 weeks old) were used for phenotypic characterization and the quantification of cell numbers. Some older mice (16 to 18 weeks old) were used in the BrdU pulse-chase assays. Mice were bred and maintained under optimal hygiene conditions in the animal facility. All experiments were performed under approved authorization within the Swiss Federal Animal Welfare Law.

Antibodies and reagents

Fluorophore-conjugated antibodies specific for CD3 (17A2), CD4 (RM4-4), CD8 (Lyt-2), CD25 (PC61), CD44 (1M7), CD6L (L-selectin, MEL14), CD24 (M1/69), CD69 (H1.2F3), CD11a (M17/4), α4β7 (DATK32), and CD29 (HMb1-1); allophycocyanin (APC)–conjugated anti-rat immunoglobulin G (IgG) and annexin V; and the chemokines CCL19 and CCL21 were obtained from BioLegend. Anti-mouse S1P1 (723412) was purchased from R&D Systems. S1P was obtained from Avanti Polar Lipids. Anti-MST1/2 pThr181/183, anti-MST1, anti-MOB1 pThr12, anti-MOB1, anti-LATS1 pSer909, anti-YAP, and anti-YAP pSer127 antibodies were purchased from Cell Signaling Technology. Anti–GFP (green fluorescent protein), anti–Hsp70 (heat shock protein 70), and anti-actin antibodies were purchased from Abcam. Anti-NDR antibodies were described previously (30). Anti-tubulin (YL1/2) and anti-Myc (9E10) were used as hybridoma supernatants. The active Rho GTPase pull-down kit was purchased from Cytoskeleton Inc., and the fold changes in the abundance of active Rho were quantified by densitometric analysis in ImageJ software.

Two-photon microscopy of popliteal lymph nodes

Fluorescently labeled wild-type, Ndr2 KO, and Ndr DKO T cells (3 × 106 to 5 × 106 cells) were injected intravenously into sex-matched C57BL/6 recipient mice, which were surgically prepared to expose the right popliteal lymph node 24 to 48 hours after adoptive transfer, as described previously (51, 52). We used the TriM Scope multiphoton microscope (LabVision BioTec) equipped with a 20× objective (numerical aperture, 0.95) for two-photon microscopic imaging. Cell migration in the T cell area of the lymph node was visualized by acquiring 12 to 16 z-stacks (in steps of 4 μm) of 150 to 200 × 150 to 200 μm xy dimensions every 20 s for 20 to 30 min. To identify the T cell area, HEVs were labeled by intravenous injection of Alexa Fluor 633–conjugated MECA-79. Velocity software (PerkinElmer) was used to transform image stack sequences into four-dimensional movies and for semiautomated tracking of cell motility. The meandering index (defined as the total displacement of a cell divided by the path length of a cell track) and average track velocity were calculated from the x, y, and z coordinates of cell centroids. Matlab was used to plot the mean displacement of a cell versus the square root of time. The motility coefficient was calculated from the slope of this graph.

BrdU labeling and detection

To quantify the proliferation of mature SP thymocytes, transition dynamics, and the emigration of mature SP thymocytes from the thymus, mice were injected twice intraperitoneally with 1 mg of BrdU within a 4-hour period, and the incorporation of BrdU into lymph nodes and thymocytes was analyzed 1, 4, 7, and 12 days later with a BrdU flow kit (BD Pharmingen) according to the manufacturer’s instructions. Briefly, a single-cell suspension was prepared from the thymus or lymph nodes. After the cells were incubated with fluorescently conjugated anti-CD4, anti-CD8, anti-CD24, anti-CD69, anti-CD25, and anti-CD62L monoclonal antibodies, the cells were fixed, permeabilized, and treated with deoxyribonuclease. The BrdU was detected with a fluorescein isothiocyanate (FITC)–conjugated anti-BrdU antibody with the BD LSRFortessa flow cytometer and analyzed with FlowJo software.

Chemotaxis assays

CD4+ and CD8+ thymocytes were sorted with a FACSAria flow cytometer to a purity >95% and then were transferred to RPMI 1640 medium supplemented with 2% FCS, 1 mM l-glutamate, 10 mM Hepes, 1 mM sodium pyruvate, and 55 μM β-mercaptoethanol. After a 1-hour incubation at 37°C, the migration of thymocytes (5 × 105 cells in 500 μl) in response to S1P or CCL19 (in a 600-μl volume) was performed in Transwell plates (5-μm pore size, Costar) according to the manufacturer’s instructions. After 3 hours, both the input population and the cells that had migrated were analyzed with a FACSCalibur flow cytometer with the same fixed speed and fixed time. The percentages of the input cells that had migrated were calculated.

Confocal microscopy

Frozen sections of thymus tissue were fixed with acetone and stained with antibodies specific for keratin 5 (Covance), fibroblast-specific ERTR7 (provided by W. van Ewijk, Utrecht, Netherlands), and CD3 (17A2), which was followed by incubation with Alexa Fluor–conjugated anti-IgG secondary antibodies (Invitrogen). Images were acquired with a Zeiss LSM700 confocal microscope (Carl Zeiss).

Cell culture, lysis, and immunoprecipitation

Mice thymi were lysed with radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS] supplemented with protease inhibitor cocktail (Roche), and equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis before being subjected to Western blotting analysis. Immunoprecipitation experiments were performed in MST interaction lysis buffer [20 mM Hepes (pH 7.4), 20 mM β-glycerophosphate, 1 mM EGTA, 2 mM NaF, 1 mM Na3VO4, 1% Triton X-100] supplemented with protease inhibitor cocktail (Roche).

T cell purification and labeling

The spleens and mesenteric and peripheral lymph nodes of wild-type, Ndr2 KO, or Ndr DKO mice were pooled and homogenized before being passed through a 70-μm cell strainer. Red blood cells were lysed by incubating the cells in 3 ml of a buffer containing tris-HCl (pH 7.5) and 0.83% ammonium chloride for 5 min. After washing, T cells were negatively selected with an EasyStep T Cell Isolation Kit (Stemcell Technologies) according to the manufacturer’s instructions. T cell purity was typically >95%. Subsequently, CD44high cells were depleted by magnetic cell sorting. We used either a purified anti-mouse CD44 antibody (BioLegend, clone IM7) that was covalently bound to Dynabeads M-450 Epoxy (Life Technologies) or a combination of biotinylated anti-mouse CD44 antibody (BD Pharmingen, clone IM7) and streptavidin-coupled RapidSpheres (Stemcell Technologies). Purified CD44low-med T cells were fluorescently labeled with 5 μM CMTMR (chloromethyl tetramethylrhodamine), 2.5 μM CFSE (carboxyfluorescein diacetate succinimidyl ester), 2.5 μM e670 (eFluor 670), or 2.5 μM CFSE and 2.5 μM e670 for 15 to 20 min at 37°C for homing assays or with 2.5 μM CFSE, 3 to 5 μM CMTMR, 2.5 μM e670, or 20 μM CMAC (7-amino-4-chloromethylcoumarin) for 20 to 25 min at 37°C for two-photon microscopy analysis. Dyes were swapped between experiments.

T cell homing

Fluorescently labeled wild-type, Ndr2 KO, or Ndr DKO T cells were intravenously injected in equal amounts (3 × 106 to 5 × 106 cells of each genotype) into sex-matched C57BL/6 recipient mice. The input mixture of cells was fixed in 1% paraformaldehyde in PBS and analyzed by flow cytometry to normalize results. Spleens, peripheral lymph nodes, mesenteric lymph nodes, and Peyer’s patches were harvested 2 hours after adoptive transfer and were homogenized and passed through a 35-μm cell strainer. Blood was collected from the vena cava with a syringe filled with PBS, 10 mM EDTA. For spleen and blood samples, lysis of erythrocytes was performed, whereas the other samples were directly washed in fluorescence-activated cell sorting buffer (PBS, 5 mM EDTA, 1% FCS). All of the flow cytometric analysis was performed with a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (Tree Star).

Statistical analysis

The Mann-Whitney test was used for the comparison of meandering index and velocity. Two-way analysis of variance (ANOVA) was used for statistical analysis of data from homing experiments. For other data, an equal variance, two-tailed Student’s t test was used to analyze the percentages of cell populations. Data in all bar graphs are means ± SD. Statistical significance was defined as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/8/397/ra100/DC1

Fig. S1. Analysis of T cell apoptosis, proliferation, and T cell subsets in wild-type and Ndr DKO mice.

Fig. S2. Analysis of thymocyte proliferation and measurement of body and spleen weights of wild-type and Ndr DKO mice.

Fig. S3. Loss of NDRs results in the accumulation of mature SP thymocytes in the perivascular space of the thymus.

Fig. S4. Loss of NDRs does not impair the homing of naïve T cells to lymphoid organs.

Fig. S5. Biochemical analysis of MST-NDR signaling in T cells.

REFERENCES AND NOTES

Acknowledgments: We thank H. Kohler for help with the flow cytometric analysis, G. Christofori and N. Hynes for support, the Friedrich Miescher Institute (FMI) Microscopy facility for help with imaging, M. Stadler for help with statistical analysis, and W. van Ewijk for the ER-TR7 antibody. Funding: F.T. and D.S.-R. were supported by the Swiss National Science Foundation SNF 31003A_138287; J.G. was supported by the Swiss Cancer League KFS 02743-02-2011; G.X. was funded by the Swiss National Science Foundation SNF 31003A_130838, and A.H. is a Wellcome Trust Research Career Development fellow (grant 090090/Z/09/Z). The FMI for Biomedical Research is supported by the Novartis Research Foundation. Author contributions: B.A.H., P.M., and F.T. conceived this project; F.T. designed the study, analyzed the data, and wrote the manuscript; J.G., X.F., J.V.S., T.B., and G.A.H. designed the experiments, analyzed the data, and participated in manuscript preparation; H.C. generated the Ndr DKO mice for preliminary analysis; D.S.-R. provided Ndr2f/f mice; D.H. helped with mouse management; D.Z. provided Mst1 KO mouse samples; L.Z., G.X., M.G., and Z.Y. helped with experiments and provided fruitful discussion; and A.H. and P.M. participated in manuscript preparation. Competing interests: The authors declare that they have no competing interests.
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