Rostral migratory stream neuroblasts turn and change directions in stereotypic patterns

Abstract

Neuroblasts generated in the adult subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Previous work uncovered motility ranging from straight to complex, but it was unclear if directional changes were stochastic or exhibited stereotypical patterns. Here, we provide the first in-depth two-photon time-lapse microscopy study of morphological and dynamic features that accompany turning and direction reversals in the RMS. We identified three specific kinds of turning (30-90 degrees): bending of the leading process proximal to the cell body (P-bending 47% of cases), bending of the distal leading process (D-bending 30%) or branching of the leading process or lamellipodium (23%). Bending and branching angles were remarkably constrained and were significantly different from one another. Cells reversed direction (>90 degrees) through D-bendings (54%), branching (11%) or de novo growth of processes from the soma (23%), but not P-bending. Direction reversal was often composed of several iterations of D-bending or branching as opposed to novel modalities. Individual neuroblasts could turn or change direction in multiple patterns suggesting that the patterns are not specific for different lineages. These findings show that neuroblasts in the RMS use a limited number of distinct and constrained modalities to turn or reverse direction.

Acknowledgements

We thank Dr. Chris Young for guidance with the Volocity software and Rosa Martinez for her help with the schematics. F.G.S. supported by NIH grant RO1 NS/AG42253-01.

Figures and Tables

Figure 1 (A–H) First frames of eight two-photon time-lapse movies used in this study with a schematic of a parasagittal section showing the SVZ/RMS pathway. The duration of each movie is indicated in the top right corner (h:min). The colored rectangles represent the location of the imaged area. Scale bar = 50 µm. (I) Schematic showing the approximate location in the RMS of the vertical limb (black arrow), elbow (red arrow) and horizontal limb (green arrow). cc, corpus callosum; ctx, cerebral cortex; dl SVZ, dorsolateral subventricular zone; LV, lateral ventricle; OB, olfactory bulb; RMS, rostral migratory stream; str, striatum. (J) Straight movement in a neuroblast exhibiting nucleokinesis. Red asterisk: stationary cell. Arrowhead shows the movement of the cytoplasmic dilation in the proximal leading process, and in the final frame, the movement of the cell body into it. Scale bar = 50 µm. (K) Schematic of cell undergoing nucleokinesis without turning.

Figure 2 Schematic showing types of turning and direction reversals. (A) P-bending is characterized by a bend in the leading process close to the soma, followed by nucleokinesis and reorientation of the cell's long axis. (B) Turning via D-bending is associated with bending of the leading process closer to the lamellipodium. (C) Branching frequently occurs at the lamellipodium and is characterized by retraction of the less dominant branch leading to a subtle shift in direction. (D) Polarity reversal involves generation of a new process on the opposite pole from the direction of migration. (E and F) Direction reversals via branching and bending are defined by successive iterations of these behaviors. Blue arrows point to the direction of migration. C, caudal; R, rostral.

Figure 3 Typical examples of turning. (A) P-bending of the leading process near the cell body. 2-dimensional frames do not show maximum angle of this cell. Arrowhead: bending introduced at the cytoplasmic dilation. Asterisk: stationary cell. Scale bar = 25 µm. (B) D-bending of the leading process. Scale bar = 25 µm. (C) Bending of the lamellipodium (arrowhead at 00:06). Scale bar = 12.5 µm. (D) Branching of the lamellipodium (arrowhead at 1:03) and retraction of the less stable branch. Scale bar = 12.5 µm.

Figure 4 Quantification of turning and direction reversals. (A) Percentage of neuroblasts turning and reversing directions in different modes. P-bending was the most common pattern for turning and D-bending of the leading process was the most common for direction reversal. (B) Duration in minutes of the different patterns for turning and direction reversal (DR). Direction reversal (DR) usually required more time than turning. Compare the means between D-bending vs. DR-bending. *p < 0.05. (C) Combinations of different patterns were observed for multiple direction changes (RD: reversing direction). Values are given as percentage of each pattern combination with respect to the total proportion of neuroblasts which exhibited multiple direction changes by combining different patterns.

Figure 5 Typical examples of direction changes. (A) Polarity reversal. Arrowhead at time point 00:30 points to the new leading process. The other arrowheads to the cell body. (B) Direction reversal via branching. The leading process rotates to come in close apposition to the cell body. Simultaneously the lamellipodium bifurcates and branches by approximately 90°. The portion pointing backwards persists. The cell body eventually translocates to the point of branching, between 01:09 and 01:12, and migration resumes. Note the bending of the leading process prior to rotation. Arrowheads point to position of cell body. (C) Direction reversal via D-bending. A neuroblast reversing direction through D-bending of the leading process (red arrow). Arrowhead shows cell soma and asterisk shows stationary cell. Scale bars = 25 µm.

Figure 6 Different morphological angles are formed by neuroblasts during turning. Cells were rotated in 3D in Volocity to reveal maximum turning angle. The angle in the leading process (A) during D-bending was measured as indicated by the superimposed angle in red. The same procedure applies to P-bending (B) and branching of the lamellipodium (C). Scale bars = 35 µm. (D) Turning patterns occurred through formation of characteristic angles by migrating neuroblasts. ***, p < 0.0005, Mann-Whitney U test between the angles formed during branching, P-bending and D-bending.

Figure 7 Distribution of turning angles in migratory versus exploratory cells. (A) Schematic illustrating the concept of turning angle ϑ_k. For simplicity we have represented it in two dimensions. However, it should be considered a turning kernel since it is defined by the x, y and z coordinates. (B–E) Distribution of turning angles for the mixed population (B), migratory (C), intermediate (D) and exploratory (E) neuroblasts. (F–H) Scatter diagrams plotting the turning angle (X axis) and the corresponding speed at that point (Y axis) in migratory (F), intermediate (G) and exploratory (H) neuroblasts.

Figure 8 Examples of individual cells exhibiting multiple turning behaviors. X, Y and Z axis show the coordinates imported to Graphis. (A) A neuroblast changing direction through multiple D-bending of the leading process (purple ellipse). The value of the average turning angle for the time points (ϑ_7–17) in which we observed this pattern is 115°. The cell's trajectory is indicated by the arrowheads. (B) A neuroblast undergoing nucleokinesis (green ellipse) followed by a direction reversal via branching (purple ellipse). Arrows point to two consecutive turning steps to achieve an orthogonal turning (coordinate x_k-1 for the calculation of ϑ₁₉ and ϑ₂₀ respectively). This is an illustrative example of the close correspondence between branch geometry (91° ≈ 90°) and the overall turning angle (50° + 45° = 95° ≈ 90°). (C) A neuroblast reversing direction via D-bending (purple ellipses) followed by nucleokinesis (green ellipse). Direction reversal occurred through two main turning steps whose values are 100° (ϑ₁₂) and 133° (ϑ₁₆). Arrows point to the x_k (nucleokinesis, ϑ₂₄) and x_k-1 (reversing direction via D-bending, ϑ₁₂) coordinate for the calculation of the turning angle. The initial point in the cell's trajectory is in the center. (D) A neuroblast undergoing nucleokinesis (green ellipse) and two iterated P-bendings (purple ellipses). The values of the average turning angle for the time points (ϑ_k-kn) in which we observed these patterns have been indicated. Arrow points to the x_k coordinate for the calculation of the turning angle ϑ₂₈. (E) A neuroblast undergoing direction reversal via D-bending (purple ellipse) followed by nucleokinesis (green ellipse). The values of the turning angle associated with these patterns have been indicated. Arrow points to the x_k coordinate for the calculation of the turning angle ϑ₉. (F) A neuroblast undergoing direction reversal via new process formation from the soma at the opposite pole of the leading process (purple ellipse). Arrow points to the x_k coordinate for the calculation of the turning angle ϑ₁₃.

Table 1 Movie features