XB-ART-14139Dev Biol 1998 Oct 15;2022:235-43. doi: 10.1006/dbio.1998.9006.
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Chondroitin sulfates modulate axon guidance in embryonic Xenopus brain.
Chondroitin sulfate proteoglycans display both inhibitory and stimulatory effects on cell adhesion and neurite outgrowth in vitro. The functional activity of these proteoglycans appears to be context specific and dependent on the presence of different chondroitin sulfate-binding molecules. Little is known about the role of chondroitin sulfate proteoglycans in the growth and guidance of axons in vivo. To address this question, we examined the effects of exogenous soluble chondroitin sulfates on the growth and guidance of axons arising from a subpopulation of neurons in the vertebrate brain which express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM. Intact brains of stage 28 Xenopus embryos were unilaterally exposed to medium containing soluble exogenous chondroitin sulfates. When exposed to chondroitin sulfate, NOC-2(+) axons within the tract of the postoptic commissure failed to follow their normal trajectory across the ventral midline via the ventral commissure in the midbrain. Instead, these axons either stalled or grew into the dorsal midbrain or continued growing longitudinally within the ventral longitudinal tract. These findings suggest that chondroitin sulfate proteoglycans indirectly modulate the growth and guidance of a subpopulation of forebrain axons by regulating either matrix-bound or cell surface cues at specific choice points within the developing vertebrate brain.
PubMed ID: 9769175
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: ncam1 noct
Antibodies: NOC-2 Ab2 Tuba4b Ab11
Article Images: [+] show captions
|FIG. 1. Immunostaining of coronal sections (A, B) and whole mounts (C–E) of stage 32 embryonic Xenopus brains. The section in A and B was double labeled for CS-56 (chondroitin sulfates containing 4- and 6-sulfates) and acetylated a-tubulin (depicts all axons). (A) Chondroitin sulfates are diffusely distributed throughout the wall of the neuroepithelium in the midbrain. Strong immunofluorescence is observed at the surface of the brain (arrows). (B) Double exposure for chondroitin sulfates in red and acetylated a-tubulin in green. Open arrows, the TPOC which is joined across the ventral midline by the VC. The strong chondroitin sulfate immunofluorescence appears to be external to the axon tracts. Embryonic brain preparations were exposed to either control medium (C) or to medium supplemented with soluble chondroitin sulfates (D–E) between stages 26 and 32. Animals were then fixed and brains imaged via confocal microscopy for acetylated a-tubulin. Control brains (C) revealed a normal distribution of axons tracts. Brains exposed to chondroitin sulfates also contained a normal complement of axon tracts; however, the trajectory of axons in the midbrain appeared more diffuse and disorganized at the junction of the TPOC, TPC, and VC (between arrowheads) in comparison to normal brains (arrowheads in C). C.S., chondroitin sulfates; DVDT, dorsoventral diencephalic tract; nPT, nucleus of presumptive telencephalon; POC, postoptic commissure; MZ, marginal zone; SOT, supraoptic tract; TPC, tract of the posterior commissure; TPOC, tract of the postoptic commissure; V, ventricle; VC, ventral commissure; VLT, ventral longitudinal tract; VZ, ventricular zone. Scale bar, 200 mm (C–E) and 80 mm (A and B).|
|FIG. 2. Schematic representation of axon tracts on the lateral surface of stage 32 embryonic Xenopus rostral brain (diencephalon and midbrain). At this stage the rostral end of the neural tube is curved about the cephalic flexure (c.f.). The dorsal surface of the diencephalon contains the epiphysis (Epi.). Although the eye vesicle is not drawn, the position of the optic stalk (o.s.) is represented. The telencephalon is not present at this age but it will emerge from the site of the presumptive telencephalic nucleus (nPT). Axons arising from this nucleus form the supraoptic tract (SOT) and contribute to the tract of the postoptic commissure (TPOC) as well as the anterior (AC) and postoptic (POC) commissures. The TPOC courses along the ventrolateral surface of the brain and at the level of the midbrain forms the ventral longitudinal tract (VLT). Axons exit the TPOC at the diencephalon– midbrain border to form the ventral commissure (VC). The epiphysis gives rise to the dorsoventral diencephalic tract (DVDT) that merges with the TPOC. Slightly caudal to the DVDT is the tract of the posterior commissure (TPC) which also fasciculates with the TPOC. The posterior commissure arises from the dorsal midbrain and crosses the midline to join the contralateral TPC.|
|FIG. 3. Confocal microscopy of NOC-21 axon tracts on the exposed lateral surface of embryonic Xenopus brain preparations. (A–F) Immunostained with NOC-2 antibodies; (G–H) double labeled with antibodies against NOC-2 (red fluorescence) and acetylated a-tubulin (green fluorescence). The NOC-21 axons in double-labeled preparations appear yellow. (A) Brain preparation exposed to unsupplemented medium exhibited a normal topography of NOC-21 axons in the rostral brain. NOC-21 axons arose from the nucleus of the presumptive telencephalon (nPT) and either crossed the midline in the postoptic commissure (POC) or grew ventrally within the supraoptic tract (SOT) and entered the ipsilateral tract of the postoptic commissure (TPOC). In the rostral midbrain, NOC-21 axons in the TPOC either turned ventrally into the ventral commissure (VC) or continued growing caudally within the ventral longitudinal tract (VLT). (B, D–F) Brain preparations exposed to medium supplemented with chondroitin sulfates. (B) The overall topography of NOC-21 axons appears similar to that observed in control animals; however, some axons have exited the TPOC and turned dorsally into inappropriate regions (filled arrows). No NOC-21 axons entered the VC (open arrows), although they continued growing into the VLT as in controls. (C) High magnification of TPOC-VC junction of a control animal clearly demonstrates the presence of NOC-21 axons in the VC. (D–F) Examples of three different brains exposed to chondroitin sulfates. No NOC-21 axons enter the VC (open arrows) while some NOC-21 axons are observed to inappropriately exit the TPOC and grow into the dorsal forebrain (filled arrows). (F) An axon inappropriately exits the TPOC and enters the ventral brain rostral to the VC. (G, H) High magnification of double-labeled brain preparations at the junction of the TPOC and VC. (G) NOC-21 axons (arrow) enter the VC and cross the ventral midline of the rostral midbrain in control brain preparations. (H) In the presence of chondroitin sulfates, NOC-21 axons fail to enter the VC despite the presence of a well formed commissure (asterisk). NOC-21 axons (arrow) continue to enter the VLT. Scale bar, 100 mm(A and B) and 50 mm(C–H).|
|FIG. 4. Effect of exposing Xenopus brains to exogenous chondroitin sulfates. Graph shows the percentage of animals that have NOC-21 axons either in the VC or in the dorsal diencephalon. It is normal for NOC-21 axons to cross in the VC and hence 100% of control animals exhibited this phenotype. It is not normal for NOC-21 axons to exit the TPOC and grow into the dorsal diencephalon and hence no axons in control animals exhibited this phenotype.|