CMW and ER conceived and designed the experiments and performed the experiments. CMW, SR, and AC analyzed the data. BS contributed reagents/materials/analysis tools. CMW and ER wrote the paper.
¤Current address: Department of Biomedical Engineering, University of California, Davis, California, United States of America
The authors have declared that no competing interests exist.
Development of many vertebrate tissues involves long-range cell migrations. In most cases, these migrations have been inferred from analysis of single time points and the migration process has not been directly observed and quantitated in real time. In the mammalian adult thymus, immature CD4+CD8+ double-positive (DP) thymocytes are found in the outer cortex, whereas after T cell antigen receptor (TCR) repertoire selection, CD4+CD8– and CD4–CD8+ single-positive (SP) thymocytes are found in the central medulla. Here we have used two-photon laser-scanning microscopy and quantitative analysis of four-dimensional cell migration data to investigate the movement of thymocytes through the cortex in real time within intact thymic lobes. We show that prior to positive selection, cortical thymocytes exhibit random walk migration. In contrast, positive selection is correlated with the appearance of a thymocyte population displaying rapid, directed migration toward the medulla. These studies provide our first glimpse into the dynamics of developmentally programmed, long-range cell migration in the mammalian thymus.
Two-photon laser-scanning microscopy reveals the change from random motion to directed migration that occurs when thymocytes undergo positive selection.
Although it is known that thymocytes relocalize from the cortex to the medulla after positive selection, the means by which this relocalization occurs is largely unknown [
In order to track migrating thymocytes in situ, we generated chimeric mice in which a fraction of thymocytes express green fluorescent protein (GFP). We devised a protocol, based on a previously described method [
Tracking software identifies the positions of individual thymocytes over time. Trajectories of individual cells are shown as tracks, which are color coded to indicate increasing time from blue (start of imaging) to yellow (end of imaging) (see
Analysis of the motility rates of individual GFP cortical thymocytes (
(A) Histogram showing the frequency distribution of average motility rates (MR) for cortical thymocytes compiled from over 1,250 tracked cells from four independently imaged thymic lobes. The vast majority of cells exhibited speeds ranging from 3 to 8 μm/min (MRlo). Approximately 7% exhibited speeds of 10 μm/min or greater (MRhi). Cells migrating between 10–13 μm/min represented approximately 5% of cortical thymocytes, and those with speeds of 14 μm/min or greater represented approximately 2% of cortical thymocytes.
(B) Instantaneous velocities versus time for representative MRhi and MRlo cells. Data are representative of 53 MRhi cells and more than 200 MRlo cells analyzed. No conversions between MRhi or MRlo behaviors were observed over a combined imaging time of more than 30 h.
(C) Five successive time frames showing the morphology associated with propulsion for an MRhi and an MRlo cell (
(D) Graph of displacement versus time for four individual MRhi and MRlo cells.
(E) Graph of directional index (Traj/D) versus average motility rate. The bars indicate the average values for Traj/D computed from 50 MRhi and 466 MRlo cells
(F) Graph of MRlo cells (left), but not MRhi cells (middle), shows linear relationship between the square of the displacement from origin versus time, indicative of random walk. Right graph shows a linear relationship between displacements from origin (as opposed to their square) with increasing time for MRhi cells, indicative of ballistic motion (right). Analysis was done on 466 MRlo cells and 50 MRhi cells from three independently imaged thymic lobes.
Further analysis showed striking differences between MRlo and MRhi cells with regard to their morphology and with other aspects of their migratory behavior. For example, MRhi cells displayed a highly polarized morphology with a well-defined leading edge and uropod, and moved by a series of lurches followed by contraction (
Examination of a cell's displacement from origin relative to time can provide additional insight into the migratory behavior of cells. Individual MRhi cells exhibit a linear relationship between displacement and time. In contrast, the displacement from origin for MRlo cells revealed numerous turns back toward cell origin (
A major aim of this study was to determine whether the localization of mature thymocytes to the medulla involves directed inward migration across the cortex, and if so, whether directed migration is a property of all cortical thymocytes or only thymocytes that have been selected to mature. To examine this question, we used graphical techniques borrowed from diffusion mechanics to distinguish movement by random walk versus directed migration [
The observation that MRhi cells moved by directed migration is consistent with the possibility that these cells are being directed to migrate toward the medulla. If this were the case, we would expect their trajectories to show a common orientation in the –
(A) Bar graph showing the average displacement in each direction by wild-type MRhi cells in a 3-min interval. Data shown were computed from 53 MRhi cells from four independently imaged thymic lobes. Data from individual runs are shown in
(B) The upper image is rotated to display the
(C) The results of step analysis (see
To confirm and extend these results, we performed a step analysis on MRhi cells (see
CD4+CD8+ double-positive (DP) thymocytes express clonally variable versions of the T cell antigen receptor (TCR). Following somatic V(D)J rearrangement and cell surface expression of the αβTCR, cortical thymocytes test out their antigen receptors for their ability to bind self-peptide and MHC proteins expressed in the thymus. A small fraction of thymocytes expressing TCR with moderate avidity for self-peptide MHC receive signals that allow them to differentiate into more mature medullary CD4+CD8– or CD4–CD8+ thymocytes, a process known as positive selection [
To test this hypothesis, we generated chimeric mice in which a small fraction (approximately 1%) of thymocytes expressed both GFP and rearranged TCR transgenes that do or do not allow positive selection. As a positive-selecting TCR, we used the class I MHC-restricted P14 TCR transgene, which promotes the development of mature CD8 T cells in the H2b (B6) background [
(A) A histogram showing the frequency distribution of average motility rates for positively selecting (blue, P14) and nonselecting (black, 5CC7) transgenic thymocytes compiled from over 1,200 P14 and 875 5CC7 thymocytes from, respectively, four and three independently imaged thymic lobes. Data (from
(B) Image showing trajectories of representative P14 MRhi cells. Note tracks for P14 thymocytes are relatively linear compared to the tracks of wild-type thymocytes (see
(C) Bar graph showing the average displacement per cell moved in each direction over a 3-min time interval (left). Data was computed from more than 100 P14 MRhi cortical thymocytes compiled from four independent experiments. Data from individual runs are shown in
(D) Results of step analysis on 412 P14 thymocytes as a function of motility rate are shown (left). Results of step analysis on 123 5CC7 thymocytes are shown for comparison (right). P14 cells moving at MR greater than 13 μm/min showed strong bias for movement in the –
Our studies show that positive selection leads to a rapid directional migration pattern and are consistent with earlier studies showing that activated CD4+CD8+ cells migrate rapidly in vitro [
As in the case of MRhi cells of wild-type mice, P14 MRhi cells displayed a highly polarized morphology, and their trajectories showed very little turning, with no incidence of pausing (
When considering thymocytes with intermediate motility rates (10–12 μm/min), there were two notable differences between P14 and wild-type thymocytes (
Importantly, we observed directional migration of MRhi thymocytes in each dataset corresponding to a region of the cortex that extends from, approximately, 80 μm to 200 μm below the thymic capsule. This suggests that thymocyte migration is directed by guidance cues that extend over a large area of the cortex. Although the nature of these guidance cues is currently unknown, there are a number of chemokines expressed in the medulla whose corresponding receptors are upregulated during positive selection [
A cortical thymocyte must travel a distance of hundreds of microns in order to reach the medulla. Based on the average distance from the capsule to the medulla in the adult mouse thymus, and the speed and directionality reported here, we estimate that a typical MRhi thymocyte that we image in the cortex could arrive at the medulla in 1 to 2 h. This short time period for migration to the medulla is in contrast to the estimates of 2–3 d for a CD4+CD8+ thymocyte to complete positive selection [
Mice expressing a GFP transgene driven by the ubiquitin promoter [
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
The 4D cell tracking was performed on 75–300 cells per movie using Imaris Bitplane software which identifies the
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).
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
(223 KB TIF).
(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
(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).
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
(76 KB TIF).
Results of displacement analyses of wild-type MRhi cells from 4 individual experiments are shown. Bar graphs show the average displacement per MRhi cell moved in each direction in a 3-min interval. Data shown were computed from 11–16 MRhi cells from each dataset. The four runs made up two separate stacks of movies (see
(73 KB TIF).
Histograms showing the frequency distribution of average motility rates for P14 cortical thymocytes were obtained from four individual runs. Compiled data are shown in
(79 KB TIF).
Results of displacement analyses of P14 MRhi cells from four individual experiments are shown. Bar graphs show the average displacement per MRhi cell in each direction in a 3-min interval. Data shown were computed from 29–35 MRhi cells from each dataset. Compiled data are shown in
(76 KB TIF).
A representative 3D image of GFP thymocytes within an intact thymic lobe. Image is rendered from one
(3.9 MB ZIP).
Time-lapse image of dataset used to generate
(2 MB ZIP).
Same dataset as shown in
(2.1 MB ZIP).
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 MRlo tracks on the right side. A small percentage of thymocytes migrate more rapidly and follow straight trajectories as exemplified by the MRhi track highlighted on the left side.
(1.5 MB ZIP).
Time-lapse image of GFP thymocytes cropped to approximately 40 × 40 × 40 μm in the
(418 KIB ZIP).
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 (
(1.4 MB ZIP).
Time-lapse image of P14 TCR transgenic GFP thymocytes in intact thymic lobe is shown rotated to display the
(1.3 MB ZIP).
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).
We thank Philippe Bousso, BJ Fowlkes, and members of Robey lab for comments on the manuscript and Holly Aaron for assistance with microscopy.
CC chemokine receptor 7
double negative (CD4–CD8–)
double-positive (CD4+ CD8+)
green fluorescent protein
motility rate
high motility rate
low motility rate
single-positive (CD4+CD8– or CD4–CD8+)
T cell receptor
three-dimensional
four-dimensional