The MST1 kinase was recently identified as playing an essential role in the promotion of lymphocyte polarization and adhesion stimulated by chemokines and TCR signaling. However, the physiological relevance of the Mst1 pathway in thymocyte development is not completely understood. In this study, we analyzed the effect of Mst1 disruption on thymocyte development and migration. Mst1-deficient (Mst1−/−) mice displayed an accumulation of mature thymocytes in the thymus, a dramatic reduction of lymphocytes in blood and peripheral lymphoid tissues, and a decrease of homing ability to peripheral lymph nodes. Mst1−/− thymocytes were impaired in chemotactic response to chemokines, such as CCL19, but not to sphingosine-1-phosphate. Further analyses of Mst1−/− mice revealed a severe impairment in the egress of mature T cells from the thymus. T lineage-specific knockout of the Mst1 gene demonstrates a cell-intrinsic role for Mst1 in regulating T cell development. Our study indicates that Mst1 is crucial in controlling lymphocyte chemotaxis and thymocyte emigration.
Thymocyte maturation follows a distinct pattern of cell migration in the thymus and ends with the egress of fully matured T cells. Chemokines and their receptors play crucial roles in regulating thymocyte migration and egress (1, 2, 3). For example, thymocyte migration from the cortex to the medulla is strictly controlled by chemokine receptor CCR7, whereas subsequent egress from the medulla to the circulation is tightly regulated by a chemoattractant lipid receptor, the sphingosine-1-phosphate (S1P)3 receptor 1 (S1P1). Disruption of the CCR7 gene (CCR7−/− mice) or of the locus encoding the CCR7-specific chemokines CCL19/21 (plt−/− mice) prevented the migration of thymocytes from the cortex to the medulla (4). Consequently, thymocytes were found trapped in the thymus of newborn CCR7−/− mice (5) and inappropriately emigrated via the corticomedullary junction in adult CCR7−/− and plt−/− mice (4). S1P1-deficient T cells develop normally but fail to leave the thymus, resulting in an accumulation of mature thymocytes in the medullar area of the thymus. Therefore, S1P1-deficient mice are lymphopenic in the periphery (6, 7).
Chemokine receptors activate multiple signaling pathways, including the Ras/Rho family of small GTPases such as Rac and Rap1 (8, 9). Lymphocytes lacking DOCK2, a Rac guanine exchange factor, show partial defects in the polymerization of F-actin induced by the S1P1 ligand S1P, as well as impaired emigration from lymph nodes (10). The actin-nucleating and polymerization protein mDia1, acting as a downstream Rho GTPase effector, is also needed for thymic egress (11). The Rap1 effector molecule, RAPL, is required for lymphocyte adhesion through LFA-1 and α4 integrins and for cell polarization triggered by chemokines (12, 13). RAPL-deficient mice display defects in thymic emigration and lymphocyte homing to peripheral lymph nodes. RAPL forms a complex with MST1 and activates MST1 kinase activity. Knockdown of Mst1 abolished the RAPL-mediated polarizing morphology and integrin-dependent lymphocyte adhesion (14).
Mst1 is a member of the Hpo pathway identified in both invertebrates and vertebrates (15, 16). Hpo, a Drosophila homologue of the mammalian Mst1 gene, controls organ size by regulating cell growth, survival, and proliferation (17, 18, 19, 20). Hpo also regulates tiling and maintenance, two complementary aspects of dendrite development in the nervous system (21). Cst-1, an Mst1 homologue in Caenorhabditis elegans, is responsible for maintenance of the normal life span and prevention of tissue aging (22). Mst1 was demonstrated in a variety of mammalian cultured cells to promote apoptosis through caspase-mediated proteolytic activation and phosphorylation (23, 24, 25) or via the Ki-Ras/Nore1 proapoptotic pathway (26). Recently, a study of Mst1 mutant mice derived from a gene trap embryonic stem (ES) cell line revealed a reduction of lymphocytes in the mutant and confirmed the role for Mst1 in the polarization and adhesion of lymphocytes upon stimulation by TCR and CCL21 (27).
In this article we report the generation and analysis of an Mst1 knockout allele that is incapable of producing any functional MST1 proteins. In addition to a severe reduction of lymphocytes in blood and peripheral lymphoid tissues, our Mst1-deficient mice displayed an accumulation of mature thymocytes in the thymus. We show that the Mst1-deficient thymocytes are impaired in chemotactic responses to chemokines such as CCL19, but not to S1P. These mice also displayed severe defects in the egress of mature T cells from thymus. A cell-intrinsic role for Mst1 in regulating T cell development is further demonstrated by T lineage-specific deletion of the Mst1 gene. Our studies reveal a novel and essential role for Mst1 in thymocyte emigration.
Materials and Methods
Generation of Mst1-deficient mice
The genomic clones containing exon 2 to exon 9 of Mst1 were isolated from a 129/Sv mouse genomic phage library (Stratagene). The Mst1 gene was modified by adding loxP sequences to XhoI and NcoI sites flanking exon 4. A neomycin (Neo) resistance cassette flanked by Flp recognition target (FRT) sites and a diphtheria toxin A (DTA) expression cassette were used as positive and negative selection markers, respectively. Gene targeting was conducted in the W4 ES cells line (ES-W4129S6; Taconic Transgenic). Chimeric mice were bred with phosphoglycerate kinase (PGK)-Flp (stock no. 003946; The Jackson Laboratory) transgenic mice with 129/Sv genetic background to remove the PGK-Neo positive selection marker and generate Mst1+/fl mice. Mst1+/− mice were derived from Mst1+/fl mice by removing exon 4 through crossing with PGK-Cre (28) transgenic mice. Progenies were genotyped by PCR containing a common oligo (Mst1-F2, 5′-GCTGATCCATGTCTCTACTCC-3′), a wild-type-specific oligo (Mst1-R2, 5′-GCTGCCATAATACTTGACTACG-3′), and a mutant-specific oligo (Mst1-R3, 5′-CCAGGCATGGTAGGGAGAATG-3′). All mice mentioned above were maintained on a 129/Sv genetic background.
To specifically mutate Mst1 gene in T cells, Mst1+/−:LCK-Cre mice were generated by crossing Mst1+/− mice with the LCK-Cre transgenic mice (29). Mst1fl/fl mice were then crossed with Mst1+/−:LCK-Cre mice on 129/Sv and C57BL/6J mixed background. Progenies were genotyped by PCR using two sets of oligos specific for Mst1 (Mst1-F2 and Mst1-R2) and Cre (Neo-Cre F1, 5′-GGAAAATGCTTCTGTCCGTTTG-3′; and Cre-R2, 5′-CGCATAACCAGTGAAACAGCATTGC-3′) genes, respectively. Six- to 9-wk-old littermates were used for all experiments described in this report. All experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of the Institute of Developmental Biology and Molecular Medicine at Fudan University, Shanghai, People’s Republic of China.
Proteins from mouse kidneys were extracted by radioimmune precipitation assay (RIPA) buffer containing 1 mM PMSF and 1× proteinase inhibitor (Roche). The proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels followed by Western blotting with anti-MST1 (Cell Signaling) as described by Callus et al. (30). The same amounts of proteins were used for the control probed with anti-actin (catalog no. sc1615; Santa Cruz Biotechnology).
Flow cytometric analysis
Single cell suspensions were prepared from thymus, bone marrow, spleen, lymph nodes (left axillary), and Peyer’s patches in PBS containing 5% FBS. Erythrocytes from blood were removed in 155 mM NH4t test.
Analysis of recent thymic emigration
The intrathymic labeling was described previously (31). Each thymus lobe of an anesthetized mouse was directly injected with 10 μl of 600 μg/ml FITC (F7250; Sigma-Aldrich) solution in PBS. Recent thymic emigration was assessed 24 h after dye injection. Spleens were harvested and analyzed for FITC+CD4+ T cells by flow cytometry. The recently emigrated thymocytes were calculated as following: FITC+CD4+ T cells in the spleen are divided by the sum of FITC+CD4+ mature T cells in the thymus and FITC+CD4+ T cells in the spleen.
Lymphocytes from WT and Mst1−/− spleen and lymph nodes were labeled with 5 μM 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR) and 1 μM 5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes), respectively, as described (32). The labeled cells were mixed with an equal number of T cells or CD4+ T cells and injected into the tail vein of 129/Sv mice. The labeled CD4+ and CD8+ T cells (or CD4+ T cells only) from the spleen, lymph nodes (axillary and brachial), mesenteric lymph node, and peripheral blood and lymph were analyzed separately by flow cytometry 1 and 24 h after injection. For the survival test, similar analyses were performed on day 5 after injection.
In vitro and organ chemotaxis assay
Organ chemotaxis assay was performed as described (34) with minor modifications; 100 μl of RPMI 1640 and 450 μl of RPMI 1640 with 500 ng/ml CCL19 were added to the upper and lower chambers, respectively. Thymocytes were allowed to emigrate out of thymus and transmigrate into the lower chamber for 3 h before subjection to Ab staining with CD4-FITC and CD8-PE and flow cytometry analysis.
Transendothelial migration assay
Assay conditions were adapted from Shulman et al. (35). bEnd.3 cells (7.5 × 104) in 200 μl of RPMI 1640 were seeded on the upper surface of a Transwell insert coated by 0.1% gelatin and cultured at 37°C in 5% CO2. Three days later the bEnd.3 cells were stimulated with 20 ng/ml TNF-α for 24 h. After washing the endothelial cell layer with PBS, thymocytes (1 × 106) in 100 μl of RPMI 1640 with 0.5% fatty acid-free BSA were added to the upper chamber, and 600 μl of medium with 100 nM S1P was added to the lower chamber. After incubation for 4 h at 37°C in 5% CO2, transmigrated thymocytes were collected to quantify the numbers by flow cytometry.
Annexin V assay
Freshly isolated splenocytes and lymphocytes were resuspended in 100 μl of binding buffer (10 mM HEPES (pH7.4), 0.14 M NaCl, and 2.5 mM CaCl2) supplied with anti-CD4 (or anti-CD8), anti-CD44 Abs, annexin V-FITC (BD Pharmingen), and 7AAD. After incubation for 15 min at room temperature in the dark, cells were supplied with 300 μl of binding buffer followed by FACS analysis within 1 h. The annexin V-positive and 7AAD-negative cells were defined as apoptotic.
Generation of Mst1-deficient mice
The Mst1 gene was mutated in ES cells via gene targeting (Fig. 1⇓A). Homologous recombinants were identified from G418-resistant colonies by PCR and then confirmed by Southern blotting (Fig. 1⇓B) using the probe shown in Fig. 1⇓A. Targeted ES cell clones were injected into C57BL/6 blastocysts to establish germline transmission of the target allele. The PGK-Neo marker and exon 4 of the Mst1 coding region were sequentially removed by crossing to the PGK-Flp and PGK-Cre transgenic lines, respectively. Deletion of exon 4 resulted in a frame shift and a premature stop codon in exon 5. WT Mst1 mRNA transcripts were not detected in the HO mutant mice by RT-PCR (Fig. 1⇓C). Western blotting showed that the MST1 protein level was reduced in HET and became undetectable in HO mice (Fig. 1⇓D). These results confirmed the disruption of the Mst1 gene by the targeted allele, which is referred as deficient or negative (−) hereafter. Animals from the Mst1+/− intercross exhibited Mendelian frequencies of inheritance. Mst1−/− mice were viable and phenotypically indistinguishable from WT and HET littermates in a 6-mo window of analysis.
A severe reduction of peripheral lymphocytes in Mst1-deficient mice
To investigate the physiological roles of Mst1 in lymphocyte development, we first examined the distribution of lymphocytes in blood and peripheral lymphoid organs in Mst1−/− mice. The average numbers of CD4+ T cells, CD8+ T cells, and B cells in blood were decreased by 85.9 ± 1.3%, 88.1 ± 5%, and 76 ± 1%, respectively, compared with the WT controls (Fig. 2⇓C). The average numbers of CD4+ and CD8+ T cells in the spleen were reduced to 45 ± 1.8% and 26 ± 0.4%, respectively, of that in WT controls. Lymphocyte paucity was also observed in axillary lymph nodes and Peyer’s patches (Fig. 2⇓D).
Mst1-deficient naive T cells are phenotypically viable
To understand the cause of lymphocyte paucity, we used an annexin V assay to assess the ongoing apoptosis of peripheral T cells freshly isolated from the Mst1-deficient mice. Because the in vivo apoptotic rates of naive T cells and activated T cells are different (36), we analyzed the apoptosis of naive and activated T cells separately. Mst1 disruption altered the proportion of naive (CD44low) and activated (CD44high) T cells in blood and secondary lymphoid organs (Fig. 3⇓A). Although activated T cells isolated from lymph nodes showed a marginal enhancement of annexin V staining, no significant alteration in ongoing apoptosis was observed among naive T cells freshly isolated from spleen and lymph nodes (Fig. 3⇓, B and C). We also assessed the effect of Mst1 deficiency on the survival of peripheral T cells in vivo in an adoptive transfer assay. Differentially labeled HO and WT T cells were cotransferred into 129/Sv hosts. The ratio of HO to WT at day 5 (0.94 ± 0.05) after transfer was essentially unchanged from that before injection (0.95 ± 0.06) (supplemental data Fig. S1).4 These results indicate that the reduction of peripheral T cells in Mst1−/− mice, particularly naive T cells, is unlikely due to a general enhancement of apoptosis.
Mst1-deficient T cells are impaired for homing to peripheral lymphoid organs
Lymphocyte adhesion and polarization play a critical role in lymphocyte homing to peripheral lymphoid organs (37). Given the reported roles of Mst1 in lymphocyte adhesion and polarization (14), we next investigated whether the homing of peripheral Mst1−/− T cells into secondary lymphoid organs was affected. Equal numbers of Mst1−/− and WT T cells labeled with the fluorescent dyes CMFDA and CMTMR, respectively, were mixed and transferred into WT recipient mice by tail vein injection. The homing ability of T cells to lymph nodes was then examined 1 h posttransfer. The ratio of labeled Mst1−/− T cells to labeled WT cells in the lymph nodes had decreased to one-half of the starting value (Fig. 4⇓B), indicating a homing defect for Mst1−/− T cells. The ratio for the two donor types in blood was found to be ∼1.5:1, which is consistent with a delayed entry of Mst1−/− T cells into lymphoid organs (Fig. 4⇓B). These results indicate that Mst1-deficient T cells are impaired for homing to peripheral lymphoid organs. We also performed similar analyses 24 h after transfer. Interestingly, it was found that the ratio of Mst1−/− to WT CD4+ T cells in the spleen was increased to ∼1.2:1. However, the ratio of Mst1−/− to WT CD4+ T cells in blood as well as in lymph was reduced to ∼0.5:1 (Fig. 4⇓C), consistent with the idea that Mst1−/− T cells are defective in emigrating into the circulation after entering secondary lymphoid organs or nonlymphoid tissues. Taken together, Mst1 is crucial for peripheral lymphocyte trafficking.
An accumulation of mature SP thymocytes in Mst1-deficient mice
We investigated whether the reduction of peripheral T cells was due to any defects in thymocyte development. Mst1-deficient mice showed normal numbers of double-positive (DP) thymocytes but a significant increase in single-positive (SP) thymocytes (Fig. 5⇓, A and B). The accumulation of SP thymocytes did not lead to any obvious perturbation of thymic cortex and medulla structures (supplemental Fig. S2), suggesting a proper occupancy of the cortex area with SP thymocytes. CD4 and CD8 SP cells in the thymus can be divided into two subgroups: CD62Llow and CD69high immature SP cells and CD62Lhigh and CD69low mature SP cells (38). Mst1-deficient SP T cell populations contained a higher proportion of mature cells with CD62Lhigh and CD69low phenotypes (Fig. 5⇓C). This result suggests that Mst1 disruption leads to an accumulation of mature SP cells in the thymus.
Mst1 is required for thymocyte egress
The accumulation of mature SP cells in the thymus and the reduction of peripheral lymphocytes in Mst1−/− mice suggests a defect in thymic egress. To examine whether Mst1−/− thymocytes are impaired in exiting the thymus, we analyzed the in vivo ability of mature thymocytes to emigrate from the thymus by tracing the recent thymus emigrants after intrathymic injection of the fluorescent dye FITC. As shown in Fig. 5⇑D, the recent emigration from the Mst1−/− thymus into the spleen was significantly less than that in WT control mice 24 h after FITC thymic injection. This result strongly suggests a defect in the output of mature T cells from the thymus to the periphery in adult Mst1−/− mice.
A T cell intrinsic role for Mst1
The above studies cannot determine whether T cell developmental defects reflect a T cell-intrinsic role of Mst1. To address this question, we generated Mst1 T cell-specific knockout mice (Mst1fl/−:LCK-Cre) by crossing Mst1fl/fl with Mst1+/−:LCK-Cre mice (supplemental Fig. S3A). Similarly as the Mst1−/− mice, Mst1fl/−:LCK-Cre mice displayed a severe reduction of peripheral T cells (supplemental data S3C) and an accumulation of SP T cells in the thymus in addition to an overall increase of the number of total thymocytes (Fig. 5⇑E). Further analyses also showed that only mature thymocytes accumulated in the thymi of Mst1fl/−:LCK-Cre mice, not immature SP thymocytes (Fig. 5⇑F). Taken together, we conclude that disruption of the Mst1 gene in mice results in a cell-intrinsic defect of thymic emigration.
Mst1-deficient thymocytes show normal chemotactic response to S1P
Egress of mature SP cells has been shown to depend on the chemotactic response of the receptor S1P1 to the chemoattractant lipid S1P (6, 7). We thus examined whether Mst1 plays a role in S1P1-mediated chemotaxis of thymocytes. As expected, immature T cells (CD4 SP, CD62Llow and CD8 SP, CD62Llow) from neither WT nor Mst1−/− mice responded to S1P (Fig. 6⇓, A and B, left panels). Mature CD4 SP (CD62Lhigh) T cells from both WT and Mst1−/− thymi displayed a similar chemotactic response to S1P (Fig. 6⇓A, right panel). Furthermore, Mst1−/− mature CD8 SP (CD62Lhigh) T cells showed a hyper-response to S1P (Fig. 6⇓B, right panel). To further evaluate the ability of Mst1−/− thymocytes to undergo transendothelial migration in response to S1P, we performed an in vitro transendothelial migration assay. The result showed that Mst1 deficiency did not influence the migration of mature T cells across endothelial cells (Fig. 6⇓, C and D). These data indicate that Mst1−/− thymocytes are capable of undergoing migration in responding to the S1P signal.
Mst1−/− thymocytes are defective in chemotaxis to chemokine CCL19, CCL21, CXCL12, and CCL25
CCL19 and CCL21 are two chemokines involved in T cell migration in thymus and thymocyte egress in neonatal mice (4, 5, 39). Knockdown Mst1 in T cells abolished CCL21-induced polarity and adhesion to ICAM (14). To determine whether Mst1 is required for CCR7-mediated thymocyte migration, we examined the chemotactic response of Mst1−/− thymocytes to CCL19 and CCL21 in chemotaxis assays. Our results showed that both Mst1−/− CD4 and CD8 SP T cells exhibited a lower response to CCL21 than WT controls (Fig. 7⇓, A and B). Mst1−/− CD4 SP T cells, but not CD8 SP cells, also showed significantly reduced response to CCL19 (Fig. 7⇓, C and D, left panels). Further analysis revealed that immature CD8 SP T cells significantly reduced the response to CCR19, whereas mature CD8 SP T cells did not (Fig. 7⇓D). To further examine whether Mst1 is directly involved in CCR7-mediated egress, we performed in vitro chemotaxis experiments using whole thymi. The study revealed that the number of mobilized thymocytes from a Mst1−/− thymus in response to CCL19 were significantly lower than that from a WT thymus (Fig. 7⇓E). These studies suggest that the CCR7-Mst1 pathway may be involved in thymocyte migration and egress.
Next, we investigated whether Mst1 plays a generic role downstream of chemokine receptors during thymocyte development. The chemokines CXCL12 and CCL25, which use CXCR4 and CCR9, respectively, as receptors, are known to be expressed in the thymus (40). CXCL12 is expressed in the medulla, the corticomedullary junction, and few cortical cells, and CCL25 is expressed in the cortex and the medulla (40). In the Transwell assay, Mst1−/− SP thymocytes displayed a significantly reduced response to CXCL12 and CCL25 (supplemental Fig. S4, A and B). Phenotyping assay showed that the expressions of CXCR4 and CCR7 on thymocytes are not affected by Mst1 knockout (supplemental Fig. 5, A and B). Collectively, these results indicate that Mst1 may play a generic role downstream of multiple chemokine receptors during thymic migration and egress.
Genetic ablation of the Mst1 gene resulted in lymphopenia. We provide compelling evidence that the lymphopenia phenotype was primarily caused by an impaired egress of mature T cells from the thymus. We further show that Mst1−/− thymocytes displayed impaired chemotactic response to several chemokines such as CCL19, CCL21, CXCL12, and CCL25. However, S1P-mediated chemotactic migration of mature thymocytes remained intact, indicating that Mst1 is unlikely to be involved in supporting S1P-induced thymic egress. These results support the notion that thymic emigration is regulated by both S1P-dependent and -independent mechanisms (41) and that Mst1 plays a particularly important role in S1P-independent chemotactic responses.
CCL19 has been demonstrated to be highly enriched on medullar endothelial venules and is speculated to mediate egress by guiding the positioning of mature thymocytes close to blood vessels (5). Recently, CCL19/CCL21 has been shown to directly activate Mst1 through engagement of CCR7 in T cells. Mst1 regulates T lymphocytes homing through induction of both a polarized morphology and integrin clustering and adhesion triggered by the ligands of CCR7, CCL19/CCL21 (14, 27). We propose that a similar mechanism could be involved in regulating thymocyte egress. This idea is supported by the observation that integrin LFA-1 expression is down-regulated in CD8 SP and mature CD4 SP T cell in Mst1−/− mice (supplemental data Fig. S6, A and B).
Although our study indicated a potential role for Mst1 in the CCR7-mediated pathway in thymocyte migration, the phenotypes of Mst1 knockout mice do not overlap with those from CCR7 knockout or plt mice. Although both CCR7 knockout and plt mice showed a dramatically reduced medulla area due to the retention of SP cells in the cortex (4), the cortex-medulla structure appears normal in Mst1−/− mice (supplemental Fig. S2). There could be multiple explanations for this difference. One possibility is that the absence of Mst1 may only reduce the efficiency of, rather than completely block, migration from cortex to medulla. A slower but constant rate of migration from the cortex to the medulla could lead to an accumulation of SP cells in the medulla if the subsequent egress is blocked. Alternatively, the loss of Mst1 function could also be partially compensated by the activity of the Mst1 homologue Mst2 (42); cortex to medulla migration may appear normal even though Mst1 is involved in regulating this process. Finally, Mst1-mediated thymic egress may be induced by several alternative chemotactic cues. Given that Mst1−/− thymocytes are defective in chemotactic responses to several chemokines in addition to CCL19, it remains to be determined which chemokine and chemokine receptor pair is directly involved in the activation of MST1 during thymic egress.
During the preparation of this manuscript, an Mst1 mutant mouse strain derived from a gene trap ES clone (AJ0315) was reported to display a reduced number of peripheral T cells (27). The reduction of peripheral lymphocytes is now recapitulated in the study of our Mst1 knockout mice. However, several phenotypes are clearly different between these two mutant alleles. First, lymphocyte reduction in the peripheral lymphoid organs was more severe in our Mst1 knockout mice than in the gene trap mutant mouse. Second, a significant increase in the number of SP thymocytes was only observed in our Mst1 knockout mice. A small but insignificant increase in SP thymocytes was mentioned in the study of the gene trap mice without additional information. Third, the study of Mst1 gene trap mice revealed a significant enhancement of apoptosis among freshly isolated peripheral CD3+ T cells (27). In contrast, analysis of our Mst1 knockout allele only showed a marginal enhancement of apoptosis among the activated (CD44high) CD4+ T cells freshly isolated from lymph nodes.
Several possibilities may account for the different results obtained in these two studies. First, these two alleles were generated by completely different methods and were structurally different. The gene trap allele carries a trapping vector (pGT0lxr; www.sanger.ac.uk/PostGenomics/genetrap/) between the first and the second exon without physical elimination of any part of the Mst1 coding sequence. Although Western blot analysis clearly demonstrated the loss of MST1 proteins in the spleen and thymus, one cannot rule out the possibility of leaky expression that may be undetectable by the method used. In contrast, our targeted allele was designed to remove the entire coding region of exon 4. This deletion will also result in a frame shift truncation if splicing occurs between exon 3 and exon 5. Therefore, the WT MST1 protein cannot be produced from our Mst1 knockout allele. Second, the discrepancy between the two studies could be partially due to the difference in genetic background between the C57BL/6 and 129Sv mouse strains. Our Mst1-deficient mice are pure 129Sv, whereas the gene trap mutant mice were progeny backcrossed with C57BL/6 for six generations. However, this background difference alone is not sufficient to explain the discrepancy between the two mutant strains, because we also observed the developmental defect among mice bred to 129Sv and C57BL/6 mixed background in the case of T lineage-specific knockout of the Mst1 gene.
Although Mst1 is broadly expressed, Mst1 disruption only results in a relatively restricted defect in lymphocyte migration and egress. Thus, the MST1 kinase and the factors in its signaling pathway represent attractive targets for the development of therapeutic drugs for immune suppression. The genetic system established in this study provides a critical tool for further delineation of the Mst1 signaling pathway relevant to thymocyte and lymphocyte migration and the development of therapeutic strategies.
During the revision of this manuscript, Katagiri, et al. reported similar findings (43).
We thank Shiyu Zhang, Boying Tan, Yanfeng Tan, Yanling Yang, and Fang Wang for technical assistance, Dr. Xiaohui Wu and Zengli Guo for scientific discussion, and Xiaofeng Tao for the editing of English.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This study was supported by Chinese Key Projects for Basic Research (973) Grant 2006CB806700, Hi-Tech Research and Development Project (863) Grant 2007AA022101, National Natural Science Foundation of China Grant 30630043, Key Projects Grant for Basic Research 08JC1400800 from the Science and Technology Committee of Shanghai Municipality, Shanghai Pujiang Program Grant 05PJ14024, and the 211 and 985 projects of the Chinese Ministry of Education.
↵2 Address correspondence and reprint requests to Dr. Wufan Tao, Institute of Developmental Biology and Molecular Medicine, Fudan University, Shanghai 200433, the People’s Republic of China. E-mail address: or Dr. Yuan Zhuang, Department of Immunology, Duke University Medical Center, Durham, NC 27701. E-mail address:
↵3 Abbreviations used in this paper: S1P, sphingosine-1-phosphate; 7AAD, 7-amino actinomycin D; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; DP, double positive; ES, embryonic stem; HET, heterozygous; HO, homozygous; PGK, phosphoglycerate kinase; S1P1, S1P receptor 1; SP, single positive; WT, wild type.
↵4 The online version of this article contains supplemental material.
- Received March 4, 2009.
- Accepted July 14, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.