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Abstract
The aim of the present study was to characterise the expression pattern of a novel N-CAM glycoform, called NOC-1, within axon tracts of the developing Xenopus brain. Double-label immunostaining of wholemount embryonic brains revealed that only a subpopulation of axons express NOC-1. Axons in the supra-optic tract (SOT) express NOC-1 and course ventrally from the presumptive telencephalon to join the tract of the post-optic commissure (TPOC), the principal longitudinal tract of the forebrain. NOC-1 was also expressed by axons that form discrete fascicles in the ventral portion of the TPOC. Growth cones in the SOT appeared to only turn caudally upon contacting the NOC-1 expressing axons in the ventral region of the TPOC. Thus, all NOC-1 expressing axons within the TPOC follow a common ventral pathway. These observations demonstrate that there is selective partitioning of axons which express a novel N-CAM glycoform within the developing Xenopus forebrain. Moreover, these results indicate that the SOT-TPOC junction is an important choice point for ventrally growing SOT axons and that selective interactions occur between subpopulations of axons expressing NOC-1 in the SOT and TPOC.
Fig1. Scaffold of axon tracts in wholemount of stage 32 embryonic Xenopus brain revealed by both singke- and double-labelling using monoclonal antibodies 6-11 and 9OE. Monoclonal antibody 6-11B-1, binds to acetylated alpjha-tubulin, which is expressed by all axons in the Xenopus brain.
A: Lateral view of a whole Xenopus brain labeled with monoclonal antibody 6-11B-1 using diaminobenzidine as a substrate to demonstrate all axon tracts and commissures, The rostral surface of the prosencephalon contains the post-optic commissure (POC). The ventral surface of the brain contains the ventral commissure (VC). All panels have similar orientations.
B: Lateral view of the whole Xenopus brain labeled with monoclonal antibody 9OE to demostrate NOC-1 expression in the subset of axons. NOC-1 is expressed by axons coursing within only the tract of the post-optic commissure (TPOC) and supra-optic tract (SOT).
C: Double-label immunofluorescence analysis at late stage 32 of acetylated-a-tubulin (green) and NOC-1 (red) using confocal microscopy. Co-expression of antigens is depicted by yellow staining. NOC-I is ex- pressed by only a subpopulation of axons within the Xenopus brain which express acetylated-a-tubulin. lndivldual images were scanned at 5 pm increments for a total of 40 pm and compiled as a single image. The white boxed area is enlarged in D.
D: High power confocal image at the junction of the TPOC and SOT showing NOC-1 expressing axons in the SOT (arrow) and in the ventral portion of the TPOC (arrowhead).
E: In early stage 32 animals, NOC-1 was expressed by a single SOT axon projecting ventrally towards the defasciculated TPOC. The growth cone of this la- beled axon (arrow) contains numerous filopodial projections.
F: Later following axons (at late stage 32) grow either individually (open arrow) or along pioneering axons (closed arrow) to form fascicles. Caudal (left); ventral (down). DVDT, dorsoventral dlencephalic tract; nPT, nucleus of the presumptive telencephalon; POC, post-optic commissure; SOT, su- pra-optic tract; TPC, tract of the posterior commissure; TPOC, tract of the post-optic commissure; VC, ventral commissure; VLT, ventral longitudlnal tract. Bar: 150 um (C), 100 um (A-B), 75 um (D), 50 um (E), 25 um (F).
Fig 2. A schematic representation of the ac=xon tracts and cell lbodies which give rise to the SOT and TPOC as previously desribed ( Chitnis ans Kuwada 1990; Wilson et al 1990; Taylor 1991; Ross et al 1992).
A: Lateral plan of Xenopus forebrain. The SOT arises from a small cluster of cell bodies with in the presumptive telencephalon, whilst the SOT arises from a small cluster of cell bodies within the presumptive telencephalon, whilst the TPOC arises from axons within both the contralateral presumptive telencephalon and the nucleus of the tract of the post-optic commissure.
B: Ros-troventral plan of Xenopus forebrain. The SOT arises from the presump- tive telencephalon, courses ventrally, and then merges with the TPOC. Axons arising from the presumptive telencephalon cross to either the contralateral TPOC via the post-optic commissure or to the contralateral presumptive telencephalon via the anterior commissure. TPOC axons originating from the contralateral presumptive telencephalon course along the ventralateral surface of the forebrain, where they are joined by TPOC axons arising from the nucleus of the tract of the post-optic com- missure. AC, anterior commissure; nTPOC, nucleus of the tract of the post-optic commissure: PT, presumptive telencephalon; POC, post-optic commissure; SOT, supra-optic tract; TPOC, tract of the post-optic commissure.
Fig. 3. Double-labeled immunoflourescence analaysis of NOC-1. ( A, C and E). and acetylated-alpha-tubulin ( B, D and F). expression at stage 32 (A-D) and stage 28 (E and F) whole Xenopus brain using opi-illumination fluorescence microscopy. A, C and E were photographed using TRITC optics and BD and F were photographed using FITC optics.
A: NOC-1 is expressed in axons in the SOT ( small arrows) and the TPOC ( large arrows) mina an early stage 32 animal.
B: The same animal as in A viewed using FlTC optics to reveal expression of acety- lated-a-tubulin in all axons. The TPOC courses as a tight bundle beneath the eye vesicle (large arrow). Caudal to the eye vesicle the TPOC splits into a number of smaller fascicles (delineated by arrowheads). Acety- lated-a-tubulin is expressed by many axons in the dorsal portion of the defasciculated TPOC, which do not c0-express NOC-1 ( compare with A).
C and D: Higher magnification of views A and B, respectively. Both axons in the SOT at this stage express NOC-1 ( thin arrows). The dashed line in C represent the dorsal limit of the TPOC ( large arrows) as determined by acetylated- alpha-tubulin expression in D. The NOC-1 positive SOT axons grow ventrally past axons in the dorsal TPOC and only turn and grow caudally after contacting NOC-1 positive axons in the ventral TPOC. E: At stage 28, prior to the formation of the SOT, NOC-1 expressing axons in the TPOC course as a single discrete bundle throughout its entire course (large arrow). The rostral surface of the prosencephalon is towards the bottom of the panel. F: Acetylated-a-tubulin expression in the same an- imal as E. There are many axons in the dorsal TPOC which do not express NOC-1 at this stage. These axons defasciculate and are present in the dorsal regions of the TPOC caudal to the eye vesicle (arrowheads). Bar: 100 pm (E-F); 50 pm (A-D).
