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Walz A
,
Anderson RB
,
Irie A
,
Chien CB
,
Holt CE
.
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Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum.
Figure 1 CS expression in the Xenopus embryonic optic pathway. Confocal micrographs of
transverse sections of stage 37/38 embryos immunostained for endogenous CSs. (A) CS immunostaining,
as visualized by the CS-56 antibody, is localized in the basal lamina surrounding the
diencephalon and to a lesser extent in the neuropil (Np) and the neuroepithelium (Ne). There is also
abundant CS expression in the mesenchyme outside the nervous system. Arrowheads represent the
area enlarged in panel E. (B–D) Specific expression of the CS subtypes. (B) Chondroitin is
expressed at higher levels in the neuropil than in the neuroepithelium. (C) Chondroitin-4-sulfate is
expressed in both the neuropil and neuroepithelium. (D) Chondroitin-6-sulfate is expressed primarily
in the neuroepithelium and at a lower degree in the neuropil. (E) A double-labeled section
showing HRP-filled RGC axons (red) growing in the CS-rich region (green) of the diencephalic
neuropil. Dorsal is up. Scale bar is 50 m in (A)–(D) and 25 m in (E).
Figure 2 The addition of exogenous CS disrupts retinal axon pathfinding. Lateral (A, B, D, E, and
F) and dorsal (C) views of stage 40 whole-mount Xenopus brains showing the trajectories of
HRP-filled RGC axons. Brains were exposed to different glycosaminoglycans during stage 33/34 to
40. (A) Control projection forms a defined optic tract (ot) in the diencephalon (Di) and correctly
enters the tectum (Tec). Black dotted curve shows the approximate border of the tectum. (B)
Projections exposed to CS (10 mg/mL) disperse widely from their normal trajectory and extend
aberrantly in the telencephalon (Tel), diencephalon, and tectum. (C) CS treated retinal axons grew
across the dorsal midline and into the contralateral tectum. White dotted line shows the dorsal
midline. (D) Disruption in retinal axon pathfinding caused by CS exposure. RGC axons show
aberrant growth in both the dorsal and ventral forebrain. (E) RGC axons exposed to heparin (100
g/mL) extend correctly through the diencephalon but veer dorsally at the tectal border and bypass
the tectum. (F) Brains exposed to keratan sulfate (10 mg/mL) have normal optic projections that
enter the tectum. Pi, pineal; dorsal is up, anterior to the left. Scale bar is 100 m in (A)–(F).
Figure 3 CS-induced pathfinding errors result in a wider
optic tract. The widths of control and CS-treated optic
projections were quantified on normalized scans of camera
lucida drawings made from wholemount brains. (A) Widths
measured at 0.1 CTU intervals (for definition of CTU refer
to Materials and Methods) show that CS treatment resulted
in a significantly wider optic tract along its entire length (p
.001 at each point). (B) Dose–response curve of CStreated
optic tracts. Tract widths were measured at mid-tract
(0.4 CTU) and show that concentrations greater than 5
mg/mL resulted in the formation of a wider optic tract.
Figure 4 CS treatment does not alter neuroepithelial organization.
Embryos were exposed to either control or CS
solutions (10 mg/mL) at stage 33/34 and examined at stage
40. (A)–(B) Confocal images of control (A) and CS (B)-
treated brain sections stained with anti-BrdU following
BrdU injections at stage 40. BrdU incorporation was restricted
to the ventricular surface (vs) of the neuroepithelium
and was unaffected by CS treatment. Exposed side is
to the left. (C)–(D) Major axon tracts were not affected by
CS treatment as seen with antiacetylated tubulin staining in
wholemount control (C) and CS-treated (D) brains. Dorsal
is up, anterior to the left. Scale bar is 100 m in (C) and (D)
and 50 m in (A) and (B).
Figure 5 Exogenous CS induces saltatory growth rates and altered growth cone morphology.
(A–D) A sequence of individual DiI labeled RGC axons growing through the diencephalon. Dorsal
is up, anterior to the left, tectum is located in the upper right corner. One axon [asterisks in (A)]
makes clear pathfinding errors [arrows in (C)] as it turns anteriorly away from the tectum.
Exogenous CS did not cause growth cone morphology to simplify. In fact, the growth cone becomes
more complex during times of stalled elongation [arrow in (D)]. (E) Axon length versus time for the
RGC axon (black squares) displayed in panels (A)–(D), compared to a control axon (open squares;
images not shown). Unlike control axons that advance at a relatively steady rate, those exposed to
CS display a saltatory growth pattern with frequent long stalling periods. (F)–(G). High magnification
confocal images of DiI-labeled growth cones in fixed preparations. (F) A control growth cone
showing an average number and length of filopodial extensions (3.5 0.3 filopodia/growth cone
and 5.8 0.4 m average filopodial length, n 67). (G) A growth cone treated with exogenous
CS shows an enlarged and more complex growth cone morphology. The number of filopodial
extensions was also increased (4.9 0.4 filopodia/growth cone and 7.2 0.5 m average filopodial
length, n 62; p .05, Student’s t test). Scale bar is 20 m in (A)–(D) and10 m in (F)–(G).
