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Although the general principles of axon guidance in vitro are understood, little is known about how axons respond to the myriad of cues in vivo and navigate axon pathways within the complex milieu of the embryonic brain. Although neuropilin-1 is an axon guidance receptor for chemorepulsive ligands in the class 3 subfamily of semaphorins, its role in directing axon growth in vivo is unknown. In the present study, we have examined the expression and role of neuropilin-1 in the embryonic forebrain of Xenopus. Neuropilin-1 was selectively expressed by a subset of axons in the early scaffold of axon tracts. These axons arise from the presumptive telencephalic nucleus, cross the rostral midline by means of the postoptic commissure, and enter the major longitudinal tract of the prosencephalon, the tract of the postoptic commissure. At the level of the mesencephalon, these axons diverge and enter one of two axon tracts: either the ventral longitudinal tract or the ventral commissure. This same population of axons also expresses NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM. We have previously revealed the presence of a chemorepulsive activity underlying the pathway followed by these axons as they cross the ventral commissure. When neuropilin-1 was overexpressed after blastomere injections of synthetic RNA transcripts, NOC-2 axons entered the ventral commissure but failed to cross the midline. Instead, these axons were inhibited from growing ventrally within the commissural pathway. These results suggest that the level of neuropilin-1 in the NOC-2 subpopulation of axons is critical for determining whether these axons reach the ventral midline. Thus, neuropilin-1 may a specific role in directing the growth of NOC-2 axons across the ventral midline in the early embryonic mesencephalon.
Fig. 1. Immunostaining of stage 32 embryonic Xenopus brains for
expression of neuropilin-1 and NOC-2. A: Confocal microscope image of
a whole-mount brain immunostained for acetylated a-tubulin to reveal the
early scaffold of axon tracts. The principal longitudinal tract of the forebrain
is the tract of the postoptic commissure (TPOC) that merges with
the ventral longitudinal tract (VLT) of the midbrain. The ventral commissure
(VC) crosses the ventral midline and connects axons to the contralateral
TPOC. The rostral surface of the forebrain contains the anterior
commissure (AC) and the postoptic commissure (POC). Axons arising
from the nucleus of the presumptive telencephalon (nPT) give rise to the
supraoptic tract (SOT) that courses ventrally to merge with the TPOC.
Two ventrally directed axon tracts also merge with the TPOC: the dorsoventral
diencephalic tract (DVDT) and the tract of the posterior commissure
(TPC). B: Whole-mount Xenopus brain immunostained for neuropilin-
1. Neuropilin-1 is expressed on a subpopulation of axons within
the early scaffold of axon tracts. C: Double-label of B with the NOC-2
antibody. Double-labelled axons appear yellow. Neuropilin-1 and NOC-2
are coexpressed by a discrete subpopulation of forebrain axons.
D: Coronal section of the Xenopus forebrain at the level of the ventral
commissure double labelled for both neuropilin-1 and NOC-2. Commissurals
axons (arrows) that cross the ventral midline express both neuropilin-
1 and NOC-2. Scale bar 5 100 mm in A�C, 50 mm in D.
Fig. 2. Overexpression of neuropilin-1 in Xenopus embryos. A: Schematic
diagram of a green fluorescent protein (GFP) injected embryo at
stage 32. The orientation of this embryo is drawn to match that presented
in Figure 1A�C. The rostral end of the brain has been rotated to show the
injected (In) green side of the embryo. The rostral midline is demarcated
by the dotted line. Green staining is also present in the ventral region of
the uninjected side of the brain due to stochastic mixing of cells at the
midline as well as to migration of axons and neural cells across the
midline. NOC-2 stained (yellow) axons arise from the injected side of the
embryo, cross the rostral midline, and enter the tract of the postoptic
commissure (TPOC) of the uninjected side. These axons either continue
growing longitudinally or turn and enter the ventral commissure. In some
embryos, NOC-2 negative axons (green) are also present in the TPOC
and ventral commissure. B: High magnification of the TPOC-ventral
commissure (VC) junction of a control embryo injected with GFP RNA
(green) and immunostained for NOC-2 (red). Double-labelled axons appear
yellow. NOC-21 axons in the TPOC (yellow) either turn and cross
the midline by means of the VC or continue growing caudally in the
ventral longitudinal tract (VLT). C�F: Embryos coinjected with GFP and
neuropilin-1 RNA exhibited disruptions of NOC-21 axons at the
TPOC-VC junction. NOC-21 axons exited the TPOC correctly, but failed
to cross the ventral midline (filled arrows), instead looping back on
themselves within the ventral commissural pathway. Some axons that do
not express NOC-1 but are mis-expressing neuropilin-1 and, hence, are
only stained green in the ventral commissure (D, F, open arrows) manage
to reach the ventral midline, whereas the NOC-2 axons overexpressing
neuropilin-1 do not. Scale bar 5 50 mm in F (applies to B�F).
