Directed migration of positively selected thymocytes visualized in real time.

Generation of GFP chimeric mice

Mice expressing a GFP transgene driven by the ubiquitin promoter [ 5 ] were used as bone marrow donors for the generation of GFP hematopoietic stem cell chimeras using a modification of a previously described procedure [ 4 ]. Whole bone marrow from a single adult GFP + mouse was aseptically harvested and resuspended into a single cell suspension in sterile Hanks' Balanced Salt Solution (Mediatech Cellgro). A total of 2–3 × 10 6 bone marrow cells were injected into newborn mice (C57Bl/6) in a volume of 70 μl. The first injection was done at 12–24 h after birth and repeated every 2–3 d for a total of four injections. Resulting chimeric mice expressed GFP in 1–2% of their thymocytes. P14 TCR transgenic mice [ 15 ] on a Rag2 –/– B6 background were obtained from Taconic. P14 +/+ Rag2 –/– mice were crossed with UBI-GFP transgenic mice to generate P14 +/- Rag2 +/– GFP +/– mice, and these mice were then intercrossed to generate P14 +/+ or +/– Rag2 –/– GFP +/+ or +/– mice. 5CC7 TCR transgenic mice [ 16 ] on a Rag2 –/– B10 background were obtained from Taconic and were crossed once with UBI-GFP transgenic mice to generate 5CC7 +/– GFP +/– Rag2 +/– mice. Bone marrow from adult double transgenic mice was used to generate chimeric mice as described above.

Two-photon imaging of intact thymic lobes Thymi from 4.5–5.5 wk-old

GFP chimeric mice were quickly harvested, lobes were separated, and the dorsal face of the lobe was adhered to 22 × 22 mm cover glass with single drop of Vetbond tissue adhesive (see Figure S2 ). Cover slip with lobe was immediately placed into a 60 × 15 mm polystyrene Petri dish containing Dulbecco's modified Eagle's medium (DMEM) without phenol red (Mediatech Cellgro). Petri dish was placed into a heated ring, and the sample was perfused with warmed media bubbled with a blend of 95% O 2 and 5% CO 2 . Sample was maintained under perfusion and held at 36.5 °C to 37.5 °C throughout imaging. Thymic lobes maintained under these conditions for up to 6 h showed no changes in cell motility and no indications of tissue deterioration. Imaging was performed as previously described [ 8 ] using an upright Zeiss NLO 510 microscope equipped with a MaiTai Ti:Sapphire laser (Spectra-Physics). For each dataset, 20–33 min of imaging was performed with the objective oriented over the top center of the thymic lobe (corresponding to the ventral side of the organ; see Figures S2 and S3 ). A total of 20 optical slices were acquired at 2-μm step intervals with a total acquisition time of 36.7 s/ z stack. Using Imaris Bitplane software, z stacks (104 × 104 × 40 μm in dimension) were processed into 3D images and reiterated through time to generate a 3D movie of thymocyte migration. In most experiments, data were acquired as “blocks” of stacked movies; a first movie was made at a maximum depth below the capsule with a second movie acquired immediately above the bottom movie (–180 to –80 μm; see Figure S3 B). Analysis of HNE-stained thymic tissue sections indicated that the area imaged invariably corresponded to cortex (data not shown). 4D data analysis The 4D cell tracking was performed on 75–300 cells per movie using Imaris Bitplane software which identifies the x, y, and z coordinates for each cell at each given time point. These statistics were exported into an Excel spreadsheet for analysis. Average motility rates (MR) were computed as the total length of migratory path divided by the total time of tracking. The extent to which a cell's migratory path deviated from a straight line was quantitated as the total length of a cell's trajectory divided by the total displacement from origin. For the determination of movement by random walk for MR lo cells, the mean-square displacement from origin was shown to be proportional to time. This relationship is given by 〈r 2 〉 = 6Dt, where r = the displacement from origin, 〈 〉 denotes the average r over numerous events at time t, and 6D is the motility coefficient [ 9 ], which characterizes the spread of cells in three dimensions. Movement by directed migration for MR hi cells was indicated by a linear relationship between mean displacement from origin (as opposed to its square) and time when computed over many events. The correlation coefficient R 2 for the best-fit line was computed in Excel using the least squares method. R 2 at p = 0.001 is statistically significant at values of 0.801 or greater. For displacement analyses of MR hi cells, the average displacement per cell in each direction was calculated from 3 min of tracking. For step analyses, thymocytes were grouped according to their average motility rates and scored as showing net displacement in the positive or negative directions along each of three axes ( x, y, and z ). The percentage of cells in each motility rate category which moved in the positive and negative directions was then graphed as a function of average motility rate. The computation of statistical significance between the frequencies of 5CC7 TCR thymocytes with high motility rates as compared to wild-type thymocytes was done in Excel using a paired t test.

Supporting Information Figure S1 Developmental Profiles of GFP+ Thymocytes from Chimeric Mice Representative profiles obtained by flow cytometric analysis of P14 and 5CC7 chimeric thymii. As expected, GFP + -gated P14 thymocytes (top row) showed high levels of TCR within the CD4 + CD8 + population and a high percentage of CD8 + SP thymocytes, indicating a high frequency of positive selection. As expected for expression of 5CC7 in a nonselecting host (bottom row), thymocytes remained arrested at the CD4 + CD8 + stage of development and fail to upregulate TCR. (128 KB TIF). Click here for additional data file.

Figure S2 Orientation of Imaging

Relative to Thymic Lobes In Vivo Thymic lobes are depicted in their normal position relative to the heart. Thymic lobes were surgically removed and separated, and then the dorsal side (side facing the heart) of thymic lobe was adhered to glass cover slip. Imaging (see Figure S3 A and S3 B) was performed with the objective positioned over the center of the ventral side of lobe. (223 KB TIF). Click here for additional data file. Figure S3 Two-Photon Imaging of Thymocyte Migration in Intact Thymic Lobes (A) Explanted GFP chimeric thymic lobe was placed in oxygen-perfused media and maintained at 37 °C throughout experiment. Objective was placed directly over the top of lobe and a total of 20 optical slices at 2-μm step intervals were acquired, which generated z stacks of 104 x 104 x 40 μm in the x, y, and z directions. The z stack acquisition was repeated every 37 s for 20–33 min. Stacks were rendered into 3D images and processed through time to yield 4D datasets (see Videos S1 – S8 ). (B) In most cases, a stack of movies was generated to increase the effective area of imaging. A bottom movie was generated by imaging starting at –160 to –200 μm below capsule and then a second movie was generated starting 2 μm above the bottom movie. (334 KB TIF). Click here for additional data file. Figure S4 Frequency Distribution of Average Motility Rates for Wild-Type Cortical Thymocytes Histograms showing the frequency distribution of average motility rates for wild-type cortical thymocytes were obtained from four individual runs. Compiled data are shown in Figure 2 A. (76 KB TIF). Click here for additional data file.

Figure S5 Displacement Analyses of Wild-Type MR hi Cells

Results of displacement analyses of wild-type MR hi cells from 4 individual experiments are shown. Bar graphs show the average displacement per MR hi cell moved in each direction in a 3-min interval. Data shown were computed from 11–16 MR hi cells from each dataset. The four runs made up two separate stacks of movies (see Materials and Methods and Figure S3 B). Compiled data are shown in Figure 3 A. (73 KB TIF). Click here for additional data file. Figure S6 Frequency Distribution of Average Motility Rates for P14 Cortical Thymocytes Histograms showing the frequency distribution of average motility rates for P14 cortical thymocytes were obtained from four individual runs. Compiled data are shown in Figure 4 A. (79 KB TIF). Click here for additional data file.

Figure S7 Displacement Analyses of P14 MR hi Cells

Results of displacement analyses of P14 MR hi cells from four individual experiments are shown. Bar graphs show the average displacement per MR hi cell in each direction in a 3-min interval. Data shown were computed from 29–35 MR hi cells from each dataset. Compiled data are shown in Figure 4 C. (76 KB TIF). Click here for additional data file. Video S1 GFP Thymocytes within an Intact Thymic Lobe A representative 3D image of GFP thymocytes within an intact thymic lobe. Image is rendered from one z stack at a single time point and is shown in a 360° rotation. Image size is 164 × 164 × 40 μm. Image was recorded approximately 140 μm below the thymic capsule. Corresponds to Figure 1 . (3.9 MB ZIP). Click here for additional data file. Video S2 Wild-Type GFP Thymocytes Migrating through an Intact Thymic Lobe Time-lapse image of dataset used to generate Video S1 . Image is shown as a maximum projection of all z stacks. Corresponds to Figure 1 . All movies were generated from 20 to 33 min of imaging and are played at six frames per second unless otherwise indicated. (2 MB ZIP). Click here for additional data file. Video S3 4D Tracking of Wild-Type GFP Thymocytes Migrating through an Intact Thymic Lobe Same dataset as shown in Video S2 with tracks highlighted. Tracks were generated using 4D cell-tracking software. The fluorescence signal from GFP thymocytes is shown in green, and the positions of individual cells as determined by tracking software are represented as grey spheres. Tracks are color coded for time from blue (start of imaging) to light yellow (end of imaging). (2.1 MB ZIP). Click here for additional data file. Video S4 4D Tracking of Wild-Type Thymocytes Reveals Distinct Migratory Behaviors A time-lapse image of GFP thymocytes in an intact thymic lobe with selected tracks highlighted. Image size is 104 × 104 × 40 μm. Note that the majority of thymocytes migrate slowly and turn frequently, as exemplified by the three MR lo tracks on the right side. A small percentage of thymocytes migrate more rapidly and follow straight trajectories as exemplified by the MR hi track highlighted on the left side. (1.5 MB ZIP). Click here for additional data file. Video S5 MR hi Cell Propulsion Is Associated with Polarized Morphology Time-lapse image of GFP thymocytes cropped to approximately 40 × 40 × 40 μm in the x, y, and z directions. Note the polarized morphology and dramatic shape changes of the MR hi cell as it crawls from bottom to upper left corner. Video shown was generated from 5 min of imaging and is played at six frames per second. Corresponds to Figure 2 C. (418 KIB ZIP). Click here for additional data file. Video S6 P14 TCR Transgenic GFP Thymocytes in an Intact Thymic Lobe Time-lapse image of P14 TCR transgenic GFP thymocytes in an intact thymic lobe. The P14 TCR induces positive selection in this system. Note that a high proportion of thymocytes migrate rapidly and in straight trajectories compared to wild-type GFP thymocytes ( Videos S2 and S3 ). Corresponds to Figure 4 B. (1.4 MB ZIP). Click here for additional data file. Video S7 P14 GFP Thymocyte Migration is Biased in the z Direction Time-lapse image of P14 TCR transgenic GFP thymocytes in intact thymic lobe is shown rotated to display the x and z dimensions. The same dataset was used to generate Video S6 . Tracks of MR hi cells are highlighted. Note that the majority of MR hi tracks are oriented in the z direction. (1.3 MB ZIP). Click here for additional data file. Video S8 5CC7 TCR Transgenic GFP Thymocytes in an Intact Thymic Lobe Time-lapse image of 5CC7 TCR transgenic GFP thymocytes in an intact thymic lobe. The 5CC7 TCR is nonselecting in this system. Note the almost complete absence of rapidly migrating thymocytes. (1.1 MB ZIP). Click here for additional data file.