QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 2, 2003
doi: 10.1242/10.1242/dev.00713

This Article Summary Full Text Full Text (PDF) Movies Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bak, M. Articles by Fraser, S. E. Search for Related Content PubMed PubMed Citation Articles by Bak, M. Articles by Fraser, S. E. Social Bookmarking                         What's this?

Axon fasciculation and differences in midline kinetics between pioneer and follower axons within commissural fascicles

Division of Biology, Biological Imaging Center, Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (87K): [in a new window]   Fig. 1. Zebrafish forebrain primary neuronal scaffold contains gata2::GFP-positive neurons. (A) 24 hpf zebrafish transmitted light view. The approximate location of the early neuronal clusters in the forebrain are schematically shown. (B) Schematic drawing illustrating the position of the major neuronal clusters and the axon tracts that connect them in the anterior CNS in zebrafish (based on Chitnis and Kuwada, 1990; Wilson et al., 1990; Ross et al., 1991). (C,D) Confocal images of dorsal (C) and lateral (D) views of a 24 hpf gata2::GFP zebrafish showing the location of GFP-positive cells in relation to forebrain morphology. The cells are located bilaterally (C) and occupy the rostroventral part of the diencephalon along the optic recess (or) (D). (E,F) Fluorescent confocal images showing the vrc stained with acetylated alpha tubulin antibody (red) to reveal the identity of gata2::GFP-expressing cells (green). White asterisk indicates a vrc cell with little or no GFP; black asterisk marks a GFP-positive vrc cell. White arrows indicate cells where colocalization of the neuronal antibody and the GFP can be seen (E). At 24 hpf all vrc cells express GFP and appear yellow due to spectral overlap between the green GFP and the neuronal antibody (F). Scale bar: 80 µm in A; 20 µm in C; 10 µm in D-F. tt, telencephalon; dc, diencephalon; drc, dorsorostral cluster; vrc, ventrorostral cluster; vcc, ventrocaudal cluster; ec, epiphysial cluster; SOT, supraoptic track; TPOC, track of postoptic commissure; POC, postoptic commissure; AC, anterior commissure; or, optic recess.

View larger version (64K): [in a new window]   Fig. 3. DiI filled gata::GFP-positive vrc cluster where first GFP axon has just started to grow to midline shows no red axons ahead of the GFP. gata::GFP embryo fixed immediately after the first GFP-positive axon began growing to midline and the vrc cluster (asterisk) was filled with DiI to label axons from the vrc. (A) Front view of the green channel showing the GFP-positive vrc cells and the first GFP-expressing growth cone (arrow) projecting towards the midline. (B) Merged green and red emission channels showing the injected vrc cluster (asterisks) and the labeled growth cone (arrow). No other DiI filled axons are visible ahead of the GFP-expressing axon. (C) Red channel showing the DiI fill and the labeled DiI growth cone (arrow). Scale bar: 10 µm.

View larger version (53K): [in a new window]   Fig. 2. gata2::GFP-positive axons pioneer the POC and also mark the majority of later POC axons. Gata2:GFP cells and their axons are depicted in green and the primary neuronal scaffold in red. Rostral is towards the left and dorsal is towards the top. Images are maximum intensity projection (MIP) views of confocal z stacks. (A-D) Lateral images at 21, 23, 25 and 27 hpf showing the relative position of gata2:GFP cells (green) with respect to the neuronal scaffold revealed with acetylated alpha tubulin (red). (E-H) Corresponding frontal views of the POC show gata2::GFP-expressing axons pioneer the POC. At 21 hpf, only early neurons differentiating away from the neuroepithelium can be seen, but no axons are present where the POC will form (broken white line) (E). At 23 hpf, a small number of axons can be seen across the POC; these early axons express GFP although because of fixation some of the GFP signal is too weak to be visible in the overlay image (F). At 25 and 27 hpf, respectively, the POC thickens. GFP-positive axons can be seen spanning the entire width of the commissure (G,H). The small number of axons that appear to not express GFP reflects axons from other brain regions that project their axons along this tract. Scale bar: 20 µm.

View larger version (90K): [in a new window]   Fig. 4. Timelapse imaging of a wild-type gata2::GFP growth cones crossing the ventral forebrain and forming the POC. (A-D) Selected images of single time points from one 3 minute interval timelapse sequence showing a typical leading growth cone (pink arrows in A and B) from the vrc cluster navigating towards and past the midline, where it is joined by the growth cone from the opposite cluster (not visible in B because it is masked by fluorescence from more dorsal sections). Subsequently, later growth cones (blue arrows in B-D) also cross the midline and grow across the POC. The midline is indicated by a broken line; time is shown in minutes. Scale bar: 10 µm. (E) A typical distance from midline along the POC trajectory axon plot. Axon length at each time point is plotted as distance from midline along the POC trajectory. The leading growth cone is plotted in pink and a later (follower) axon is plotted in blue corresponding to the growth cones marked with pink and arrows in A-D. The axons shown here start from the right vrc (top of the plot) and cross over to the left vrc (bottom of the plot) as indicated in the schematic to the left of the graph. The two axons advance across the midline with different rates as indicated by the slope of each line (see pink and blue boxed regions). On the right, two microscope configurations show how the imaging was performed. The timelapse shown in A-D was obtained in the inverted configuration.

View larger version (26K): [in a new window]   Fig. 5. POC axon dynamics with respect to the midline. Quantitative analysis of axon growth rates reveals a difference in axon behavior around the midline not readily apparent from direct observation of timelapse experiments. (A,B) Representative plots of distance from the midline along the POC trajectory versus time for leader (A) and follower (B) axons. Leader axons spend longer time within ±10µm of midline (broken black line in A-C), while the growth of later axons does not slow down in this region. (C) Average behavior of leader (n=16) and follower (n=24) axons at the midline. Leader and follower axon plots were centered on the timepoint when each axon crossed the midline. The average plot shows a more than twofold difference between the time leader and follower axons stay at the midline. Individual plots of all axons from the left vrc were reflected around the x-axis. (D) Average growth rates±s.e.m. for leader (pink) and follower (blue) axons with respect to the midline and outside midline region. Leading axons grow significantly more slowly at the midline compared with their average growth rate away from midline and compared with follower axons at the midline (P<0.001, Student's t-test corrected for multiple comparison).

View larger version (73K): [in a new window]   Fig. 6. Growth cone morphology analysis for POC axons. Growth cone morphology can be visualized during timelapse imaging of the POC axons. (A-C) Representative growth cones of leader (long pink arrow) and follower (long blue arrow) axons are shown. Filopodia present on both types of growth cones are also indicated with smaller arrows (pink for leader and blue for follower). Scale bar: 10 µm. (D) Average growth cone areas do not differ between leader (n=12) and follower (n=8) axons. Only clearly visible follower axons were chosen for this analysis. (E) Average width to length (w/l) ratio plot shows a significantly higher ratio for leader axons (n=12) compared with follower axons (n=8) (P<0.05, Student's t-test).

View larger version (67K): [in a new window]   Fig. 7. In the absence of a leader axon, follower axons grow more slowly at the midline. (A) Single time point images showing a leader axon (pink arrow) that projects towards the midline and is injured right before crossing it (pink arrow with asterisk). A follower axon behind it (blue arrow) overtakes the leader axon and crosses the midline (blue arrow with asterisk). After this axon crosses the midline another axon from the left vrc also grows across the midline. The two growth cones: the new leader from the right vrc (blue arrow with asterisk) and the first axon from the left vrc have just undergone their stereotypic behavior and are not aligned with the POC trajectory until the last image where their growth cones establish contact with the opposite axon shaft. (B) Axon distance from midline along the POC trajectory graph showing three separate cases where follower axon was analyzed after the leader axon was damaged. The axon graphed in blue corresponds to the follower axon shown above (blue arrow in A).

View larger version (56K): [in a new window]   Fig. 8. Leader axon alters midline kinetics of first contralateral axon at the midline during commissure formation. Single time point images showing stereotypic interaction between the initial POC growth cones. Two growth cones, one from each side, are visible with one already at the midline (left). Up to this point both growth cones have complex morphologies. Upon contact, however (middle panel), both growth cones change shape and become elongated (right) even though the second growth cone has not yet crossed the midline. When this second axon grows across, it has fast midline kinetics similar to a follower axon. Scale bar: 10 µm.

View larger version (36K): [in a new window]   Fig. 9. How fasciculation between commissural axons alters their midline kinetics. A schematic drawing shows a leader axon (pink) and a number of follower axons (blue and black) growing through the midline. The leading axon being the first, is completely exposed to the guidance cues in the environment. Its growth cone must sense all the positive and negative midline cues and interpret them accordingly, which results in slow kinetics of leader axons at the midline where these cues are found. By growing along the leader, follower axons are less exposed to midline cues. This can happen because their growth cones are shaped differently, which limits their exposure to conflicting midline signals and/or because the substrate that the leader axon provides contributes an extra signal that allows them to grow across the midline swiftly.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter