(a) Representative FACS analysis of CD4 and CD8 expression on thymocytes from DO11.10+ WT and DO11.10+ GIT2^−/− mice to assess stages of thymocyte development. Numbers in each quadrant represent the percentage of cells in the indicated quadrant. (b) Quantification of cell numbers of different thymic subsets from DO11.10+ WT or DO11.10+ GIT2^−/− mice. Error bars: s.e.m. (n=10–11 mice per genotype). (c) Expression of TCR transgene assessed by transgene specific antibody (KJ1-26) in total thymocytes from DO11.10+ WT or DO11.10+ GIT2^−/− mice. (d) Percent of KJ1-26^hi CD69^hi cells in DP thymocytes and KJ1-26^hi CD69^hi DP cell number from DO11.10+ WT or DO11.10+GIT2^−/− mice. Error bars: s.e.m. (n=3 mice per genotype). (e) Expression of KJ1-26 in CD4 T cells from DO11.10+ WT or DO11.10+ GIT2^−/− spleen. (f) Percent of CD4⁺ KJ1-26⁺ cells in the periphery. (e,f) Graphs in this figure show mean ± s.e.m. (DO11.10+ WT mice, n=3; DO11.10+ GIT2^−/− mice, n=5) *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001 (unpaired two-tailed Student’s t-test).

(a) FACS analysis of WT BALB/c hosts reconstituted with DO11.10+WT or DO11.10+GIT2^−/− donor bone marrow showing the defect in CD4 SP generation from DO11.10+GIT2^−/− is hematopoietic cell intrinsic. Results are representative of three independent experiments. (b) FACS analysis of chimeras, in which WT and GIT2^−/− hosts were reconstituted with DO11.10+WT bone marrow. Results are representative of five chimeras for each condition. (c) Thymic architecture of chimeras that were reconstituted with DO11.10+WT or DO11.10+GIT2^−/− bone marrow cells. (d) One to one mixed bone marrow chimera using two different donor groups with different congenic markers (OTII+WT (CD45.1/CD45.2) and OTII+GIT2^−/− (CD45.2/CD45.2)) revealed the defect in DP to CD4 SP transition in thymocytes generated from OTII+GIT2^−/− bone marrow hematopoietic stem cells (HSCs). Results are pooled from seven independent experiments. Graphs in this figure show mean ± s.e.m. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001 (unpaired two-tailed Student’s t-test).

(a) Transwell migration assays were performed with thymocytes in the upper well in the absence of SDF-1 (media) and in the presence of SDF-1 in the top well (Top), in the bottom well (Bottom), and in both top and bottom wells (Both) using WT and GIT2^−/− thymcoytes. Results are representative of two independent experiments. (b) Directional migration of WT and GIT2^−/− thymocytes on two-dimensional surfaces was monitored using SDF-1 releasing alginate beads and time-lapse fluorescence microscopy. WT and GIT2^−/− thymocytes were labeled with CFSE (0.2 μM) and CMTMR (2 μM) respectively. A 1:1 mixture of thymocytes mixed plus 1.5 mg/ml collagen were placed on ICAM-1 (1 μg/ml) coated chambered coverglass in the presence of non-loaded (control) or SDF-1- loaded beads. Motility of WT (CFSE) and GIT2^−/− (CMTMR) thymocytes were imaged for 40 min to 1 hour at 30 second intervals. Cell movement was measured based on the center of mass of GFP or CMTMR fluorescence using Imaris tracking software (Bitplane). To compare the average migration speed (path length/time) of WT and GIT2^−/− thymocytes in the presence of control beads or SDF-1-loaded beads, the average migration speed was calculated from cells based on two criteria. The first criterion required a displacement of more than 20 μm (about twice of cell length). The second criterion required the cell to be in the field for more than 500 sec if it did not move more than 20 μm. By using both criteria, almost all cells in the field in both conditions (beads only or SDF-1-loaded beads) were included for the analysis. Each dot represents a track of a thymocyte. Data are representative of two (control beads) and three (SDF-1 releasing beads) independent experiments. Graphs in this figure show mean ± s.e.m. (n.s.: not significant; *: P=0.03 (unpaired two-tailed Student’s t-test)). (c) The average migration speed of WT and GIT2^−/− thymocytes in the presence of control beads or SDF-1-loaded beads shown in (b) is binned into 1 μm/min intervals. Percentages of total cells that migrate within the particular range were measured and then normalized as a percentage of maximum (% of Max). (d) Left panel: The directionality index of WT and GIT2^−/− thymocytes that migrate more than 20 μm towards SDF-1 releasing beads. Directionality index was determined by dividing displacement from origin by path length (n.s.: not significant, unpaired two-tailed Student’s t-test). Right panel: To determine directional motility, the average migration speeds of WT and GIT2^−/− thymocytes that moved towards SDF-1 releasing beads by more than 20 μm were binned into 1 μm/min intervals. For this analysis, cells that did not move towards SDF-1 releasing beads by more than 20 μm are excluded. Percentages of total cells that migrate within the particular range was measured and then normalized as a percentage of maximum (% of Max). Data are representative of three independent experiments.

(a) Filters of transwells were coated with either 100μl of PBS alone or PBS containing ICAM-1 (3 μg/ml). Migration activity of WT or GIT2^−/− thymocytes in the absence or presence of SDF-1 was measured using BSA- or ICAM-1- coated filters. Migrated thymocytes were collected, stained with anti-CD4 and CD8, and quantified using flow cytometry. Migratory activity of DP population is presented as a percentage of migrating DP cell number divided by input DP cell number. (b) Filters of transwells were coated with 100 μl of PBS containing ICAM-1 (3 μg/ml) plus two concentrations of CD3 (5 μg/ml or 20 μg/ml). Migratory activity of WT or GIT2^−/− thymocytes in the absence or presence of SDF-1 was measured. Data are from two independent experiments with duplicates per condition (error bars: s.e.m.). *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001 (unpaired two-tailed Student’s t-test).

(a) Intracellular staining of BrdU⁺ cells after 48 hours of BrdU incorporation using TCR transgenic WT or GIT2^−/− mice. Thymocytes were labeled continuously with BrdU, as described in the Methods section. BrdU incorporation within the DP or CD4 SP thymocytes is plotted as a percentage of total live thymocytes (error bars: s.e.m.). Eight mice for OTII+ WT, six mice for OTII+ GIT2^−/−, three mice for DO11.10+ WT or DO11.10+ GIT2^−/− were used. (b) Apoptosis assay using 7AAD/Annexin V staining at 0, 4, 24 hours of incubation in 10% FBS containing media in DO11.10+WT or DO11.10+ GIT2^−/− thymocytes. Data are representative of two independent experiments using two or three mice per genotype for each experiment. (c) Intracellular staining of active caspase 3 in DO11.10+WT or GIT2^−/− thymocytes. Data are pooled from two independent experiments using five mice for each genotype. (d) Percentages of Vβ3, Vβ5 and Vβ11 positive cells in CD4 SP thymocytes or CD4+ T cells from lymph nodes from BALB/c background WT and GIT2^−/− mice are shown (error bars: s.e.m.; n=3 per each genotype). *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001 (unpaired two-tailed Student’s t-test).

(a) Transwell migration assays were performed in the absence or presence of SDF-1 or CCL25 using WT and GIT2^−/− thymocytes. Migrated thymocytes were collected, stained with anti-CD4 and CD8, and quantified using flow cytometry. Migration activity of distinct populations is presented as the percentage of input cells that migrated to the lower chamber. Bars indicate means and the standard errors of the means (s.e.m.) are indicated. Data are from five independent experiments with duplicates per condition. (b) Representative FACS analysis of CD69 and TCRβ expression (upper panels) or CD69 expression (lower panels) in DP populations of input or migrated thymocytes from WT or GIT2^−/− mice in the presence of CCL25 or SDF-1. Migrated thymocytes were collected, stained with anti-CD4, CD8, CD69 and TCRβ, and quantified using flow cytometry. (c) Migration of pre-selection CD69^lowTCRβ^low (left) and post-selection CD69^hiTCRβ^hi DP thymocytes (right) are presented as a percent of input. Data are from three independent experiments with duplicates per condition (error bars: s.e.m.). *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001 (unpaired two-tailed Student’s t-test).

(a) Increased activation of Rac1 after 0, 0.5, 2, 5, 15 min of SDF-1 stimulation using WT or GIT2^−/− thymocytes (left) WCL; whole cell lysates; Fold increase in Rac1 activation normalized to GTP-Rac1 in the resting state (right). (b) Increased actin polymerization in response to SDF-1 in GIT2^−/− thymocytes as assessed by phalloidin-Alexa488, CD4 and CD8 staining. The mean fluorescence intensity of phalloidin-Alexa488 was measured in the DP (left) and CD4 SP (right) thymocyte populations and normalized to the value of WT at rest. Data are from three independent experiments per condition (error bars: s.e.m.).

(a) Schematic diagram of the experimental set-up. Bone marrow cells from GIT2^−/− mice expressing a GFP transgene under the control of the ubiquitin promoter (UBI-GFP) and WT mice expressing a CFP transgene under the control of actin promoter (Actin-CFP) were mixed with WT bone marrow cells, then transferred to irradiated hosts to generate partial hematopoietic chimeras in which about 1% of thymocytes are derived from the GFP-or CFP- expressing donor. After 6–8 weeks, intact thymic lobes from adult chimeric mice were imaged using TPLSM. (b) Upper panel: CFP (WT, blue) and GFP (GIT2^−/−, green) thymocyte images from thymi of a partial mixed bone marrow chimera at a single time point superimposed on cell tracks. Red: blood vessels. Lower panel: Trajectories of individual cells are shown as tracks. Yellow (CFP+ WT thymocytes), Purple (GFP+ GIT2^−/− thymocytes). (c) Average migration speed (path length/time) of WT and GIT2^−/− thymocytes. Each dot represents an individual tracked thymocytes. Data are compiled from one run for WT and three runs on two different days of imaging for GIT2^−/−. ***: P<0.0001 (unpaired two-tailed Student’s t-test). (d) Directionality index (displacement/path length) plotted as a function of average speed (0–10 μm/min) of WT and GIT2^−/− thymocytes. Average speeds of cells were binned between 2–3, 3–4 and 4–5 μm/min (grey line) and average directionality index of each bin is indicated (***, P=0.0004 for 2–3 μm/min speed; ***, P<0.0001 for 3–4 μm/min speed; ***, P<0.0001 for 4–5 μm/min speed, unpaired two-tailed Student’s t-test). (e) Example of GIT2^−/− thymocytes migrating near blood vessels. Fluorescent image is a projection of single time point with cell tracks superimposed. (f) Immunofluorescence of anti-SDF-1 staining in the thymus, showing SDF-1 positive blood vessels. Thymic sections injected with lectin-FITC to label blood vessels were stained for SDF-1 (Upper panel: 20x, Lower panel: 40x magnification).