Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Anderson RB
,
Cooper HM
,
Jackson SC
,
Seaman C
,
Key B
.
???displayArticle.abstract???
In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventralmidbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventralmidbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain.
FIG. 1. Immunostaining of stage 32 embryonic Xenopus brain. (A) A lateral view of a Xenopus brain double labeled for acetylated
a-tubulin (green fluorescence) and NOC-2 (red fluorescence). Double-stained axons appear yellow. These axons course in the ventral region
of the tract of the postoptic commissure (TPOC). Many NOC-21 axons cross the midline in the ventral commissure (VC) while some
continue growing caudally in the ventral longitudinal tract. Rostral is to the left. The ventral surface of the brain contains the ventral
commissure. (B) Schematic representation of the trajectory of axons in the principal axon tracts in the Xenopus brain. (C) Cross section of
the rostral Xenopus brain at the level of the ventral commissure labeled for DCC. Strong staining was observed in the TPOC (arrows) as
well as in axons exiting this pathway and crossing the ventral midline. (D) The same section as in C labeled also for NOC-2 (red
fluorescence). NOC-21 axons that also express DCC appear yellow. All NOC-21 axons in the TPOC (unfilled arrows) and ventral
commissure (arrowhead) are yellow and hence coexpress DCC. AC, anterior commissure; DVDT, dorsoventral diencephalic tract; nPT,
nucleus of the presumptive telencephalon; PC, posterior commissure; POC, postoptic commissure; SOT, supraoptic tract; TPC, tract of the
posterior commissure; TPOC, tract of the postoptic commissure; VC, ventral commissure of the tegmentum; VLT, ventral longitudinal
tract. Scale bar, 100 mm in A; 50 mm in C and D.
FIG. 2. (A) Schematic diagram of the soluble subdomain of mouse
DCC (mDCCFn-2). Comparison of the amino acid sequences of
mouse and Xenopus DCC reveals that these orthologues are highly
conserved as indicated. (B) The purity of the mDCCFn-2 protein
preparation was demonstrated by PAGE analysis followed by silver
staining. (C) The identity of the purified mDCCFn-2 protein was
confirmed by Western blot analysis using a polyclonal antipeptide
antiserum raised against the first 20 amino acids of the mature
mouse DCC protein. This antiserum does not cross-react with the
unrelated FLAG-tagged protein, VEGFR2-EX.
FIG. 3. Effects of culturing exposed Xenopus brain preparations with either recombinant soluble DCC or control proteins. (A–C) Whole
mounts of embryonic Xenopus brains cultured in either unsupplemented medium (A) or medium supplemented with VEGFR2-EX (B) or
mDCCFn-2 (C) developed normally when immunostained for acetylated a-tubulin. (D–F) Brain exposed to VEGFR2-EX and double labeled
for acetylated a-tubulin (green fluorescence) (D) and NOC-2 (red fluorescence) (E). D and E are scans for a single fluorophore while F is a
composite of double-labeled images in E and F. NOC-21 axons (arrow) exit the TPOC and enter the VC where they cross the midline. (G–I)
Brain exposed to the recombinant soluble DCC receptor (mDCCFn-2) and double labeled for acetylated a-tubulin (green fluorescence) (G)
and NOC-2 (red fluorescence) (H). G and H are scans for a single fluorophore while I is a composite of double-labeled images in H and I.
Axons at the junction of the TPOC and the VC appear disorganized (asterisk). NOC-21 axons (arrowheads) clearly form whorls at the
junction of these two tracts. (J–L) Examples of NOC-21 axons at the junction of the TPOC and VC in three different brain preparations
exposed to the recombinant soluble DCC. Many axons stalled in the ventral commissural pathway after exiting the TPOC (unfilled arrows)
(J and K). Some axons extended ventrally into the VC but then turned about and coursed dorsally (arrowheads) (L). Axons were also observed
to exit the TPOC inappropriately (filled arrows) (K and L). Scale bar, 150 mm in A–C; 50 mm in D–L.
FIG. 4. Xenopus brain preparations exposed to a DCC-blocking monoclonal antibody (x-DCC). Many NOC-21 axons extended ventrally
into the VC but then either stalled or turned about (arrowheads) (A and B). Some axons clearly exited the VC and grew back toward the
TPOC (unfilled arrow) (A). Scale bar, 50 mm in A and B
FIG. 5. Phenotypic effects of expression of dominant negative DCC (A–C) in Xenopus embryos. (A) In vitro-transcribed dominant negative
(dom2ve) DCC and GFP-capped cRNAs were co-injected into single blastomeres at the two- to four-cell stage. The dom2ve DCC lacked the
cytoplasmic tail present in wild-type DCC. Animals were allowed to survive until stage 32 and whole mounts of brains were then
immunostained for NOC-2 (red fluorescence). NOC-21 axons expressing dom2ve DCC were labeled yellow, as they coexpressed GFP. (B)
Control animal injected with GFP alone demonstrated that yellow axons entered the ventral commissure normally. (C) NOC-21 axons
expressing dom2ve DCC (yellow axons) exited the TPOC but failed to cross the midline. Instead these axons turned about and grew back
dorsally (arrowheads). Scale bar, 50 mm in B and C.
