© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
RESEARCH ARTICLE
NRP1-mediated Sema3A signals coordinate laminar formation in
the developing chick optic tectum
ABSTRACT
The optic tectum comprises multiple layers, which are formed by radial
and tangential migration during development. Here, we report that
Neuropilin 1 (NRP1)-mediated Sema3A signals are involved in the
process of tectal laminar formation, which is elaborated by tangential
migration. In the developing chick tectum, NRP1, a receptor for
Sema3A, is expressed in microtubule-associated protein 2 (MAP2)positive intermediate layers IV and V. Sema3A itself is a diffusible
guidance factor and is expressed in the overlying layer VI. Using stable
fluorescent labeling of tectal cells, we show that MAP2-positive
intermediate layers are formed by the neurons that have been
dispersed by tangential migration along the tectal efferent axons.
When Sema3A was mis-expressed during laminar formation, local
Sema3A repelled the tangential migrants, thus eliminating MAP2positive neurons that expressed NRP1. Furthermore, in the absence of
the MAP2-positive neurons, tectal layers were disorganized into an
undulated form, indicating that MAP2-positive intermediate layers
are required for proper laminar formation. These results suggest that
NRP1-mediated Sema3A signals provide repulsive signals for
MAP2-positive neurons to segregate tectal layers, which is important
in order to coordinate laminar organization of the optic tectum.
KEY WORDS: Laminar formation, Optic tectum, Sema3A, NRP1,
Cell migration, Chick
INTRODUCTION
The optic tectum is the main visual center for non-mammalian
vertebrates. The avian optic tectum comprises 16 laminae, which are
subdivided by distinct neuronal cell types (Ramón y Cajal, 1911;
La Vail and Cowan, 1971a; Senut and Alvarado-Mallart, 1986). In
chick embryos, the dorsal part of the mesencephalon expands after
embryonic day 3 (E3) through massive cell proliferation to form
tectal swelling. After proliferating in the ventricular layer, postmitotic neuronal precursor cells migrate radially to their destinations
(Gray et al., 1988; Gray and Sanes, 1991). The process of layer
formation has been extensively studied in the mammalian cerebral
cortex, which comprises six layers in which post-mitotic cells
migrate in an inside-out type manner (Angevine and Sidman, 1961;
Rakic, 1974; McConnell, 1988). The mode of migration of tectal
post-mitotic cells is very different from that of the cerebral cortex.
Three waves of radial migration take place during laminar formation
of the chick optic tectum. The first migration between E3 and E5
1
Department of Molecular Neurobiology, Graduate School of Life Sciences, Tohoku
2
University, Aoba-ku, Sendai 980-8575, Japan. Institute of Development, Aging &
3
Cancer, Tohoku University, Sendai 980-8575, Japan. Department of
Neuroanatomy and Embryology, School of Medicine, Fukushima Medical
University, Fukushima 960-1295, Japan.
*Author for correspondence ([email protected])
Received 17 March 2014; Accepted 27 July 2014
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forms the inner laminae, the second migration between E4 and
E7 forms the outer laminae and the third migration between E5
and E8 forms the middle laminae (La Vail and Cowan, 1971b;
Sugiyama and Nakamura, 2003). In addition to radial migration,
tangential migration also occurs during tectal laminar formation
(Domesick and Morest, 1977; Puelles and Bendala, 1978). After E6,
some radially migrating cells change direction to a tangential route
and migrate dorsoventrally to differentiate into multipolar neurons
(Gray and Sanes, 1991; Martínez et al., 1992). These observations
suggest that tangential migration might be a key process for building
tectal architecture, as in the cerebral cortex (Marín and Rubenstein,
2001). However, how tangential migration and its derivative cells
are implicated in the laminar formation of the optic tectum remains
elusive.
The neuropilin-semaphorin system plays diverse roles in
development, including the regulation of cell migration (Tran
et al., 2007). Class 3 secreted semaphorins direct the migration of
various cell types, such as oligodendrocytes (Spassky et al., 2002),
neural crest cells (Eickholt et al., 1999), endothelial cells (Miao
et al., 1999; Serini et al., 2003; Shoji et al., 2003) and leukocytes
(Delaire et al., 2001). Semaphorin-mediated guidance is
bi-functional as it serves as an attractant or repellent depending
upon the cell type. In laminar formation of the cerebral cortex,
Sema3A attracts cortical neurons expressing Neuropilin 1 (NRP1),
a receptor for class 3 semaphorins, during radial migration of the
cortical neurons (Chen et al., 2008). By contrast, Sema3A and
Sema3F repel cortical interneurons that express NRP1 and/or
NRP2 receptors, preventing tangential migration of these cells
into the striatum, instead, channeling them into the cortex (Marín
et al., 2001).
In the developing chick optic tectum, it has been reported that
NRP1 is expressed in specific tectal layers that have a binding
affinity with secreted Sema3A (Takagi et al., 1995; Feiner et al.,
1997). We thus questioned whether NRP1-mediated Sema3A
signals play a role in tectal laminar formation. In our preliminary
study, we noticed that the tectal cells in the intermediate layers
express NRP1 and that these cells are derived from the tangentially
migrating cells. These observations suggested that tangential
migration might contribute to the process of laminar formation,
which might be regulated by NRP1-mediated Sema3A signals.
In the present study, we aimed to assess the contribution of
tangential migration to tectal layer formation and to examine the role
of NRP1-mediated Sema3A signals in this process. We observed the
migration of tectal post-mitotic cells through stable labeling of tectal
cells with fluorescent proteins and demonstrated that tangential
migration occurred along the tectal efferent axons. The cells after
migration differentiated into multipolar neurons in layers IV and V
(layers IV/V), which expressed MAP2 and NRP1. Mis-expression
of Sema3A eliminated the multipolar neurons that expressed NRP1,
resulting in the undulation of the overlying layer VI. We concluded
that NRP1-mediated Sema3A signals play a role in the formation of
DEVELOPMENT
Yuji Watanabe1,2,3,*, Chie Sakuma3 and Hiroyuki Yaginuma3
RESEARCH ARTICLE
the intermediate layers IV/V which is important to coordinate
laminar organization of the optic tectum.
RESULTS
Sema3A and NRP1 are expressed in distinct tectal layers
It has been reported that NRP1 is expressed in the specific layers in
the developing chick optic tectum (Takagi et al., 1995). Staining
with an NRP1-specific antibody revealed that NRP1 was expressed
in layers IV/V at E8.5 (Fig. 1A-C, roman numerals indicate tectal
layer number; Yamagata et al., 2006; La Vail and Cowan, 1971a).
We found that microtubule-associated protein 2 (MAP2), a neuronal
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
marker, was also expressed in layers IV/V (Fig. 1D-F; Yasuda and
Fujita, 2003). Expression of Sema3A, a ligand for NRP1, was
localized in layer VI (Fig. 1G,H). The protein localization of
Sema3A was examined through binding to chick NRP1-AP, which
contains extracellular domains of the receptor fused with alkaline
phosphatase. Although NRP1-AP was immobilized to layer VI
(Fig. 1K,L), a control alkaline phosphatase protein did not bind to
any tectal layers (Fig. 1M,N). Expression of Sema3A mRNA and
NRP1-AP binding suggest that the Sema3A protein is localized to
layer VI, which overlays NRP1-expressing layers IV/V.
MAP2-positive neuronal layers are formed by the tangential
migrants
Fig. 1. Sema3A and NRP1 are expressed in distinct tectal layers. Chick
optic tectum at E8.5 sectioned perpendicular to the anterior-posterior axis of
the tectum. (A-C) Staining with a NRP1-specific antibody revealed that NRP1
is expressed in layers IV/V (B,C), which cover the whole tectal area along the
dorsoventral axis (A; lower magnification). (D-F) MAP2 is also expressed in
layers IV/V (E,F) and in the whole tectum (D; lower magnification).
(G-J) Detection of Sema3A mRNA using an anti-sense (-AS) probe (G,H) or a
sense (-S) probe (I,J). (K,L) NRP1 extracellular domain tagged with alkaline
phosphatase (NRP1-AP) was used to detect the protein localization of
Sema3A. NRP1-AP binds to layer VI. (M,N) The control alkaline phosphatase
protein does not bind to any tectal layers. In layer discrimination, layer VI is
distinguished by a high cell density, which is revealed through DAPI staining.
Layer IV and V can be distinguished by the difference in nuclear density and
also in cytoskeletal organization after MAP2-staining (see Fig. 2I). Scale bars:
1 mm (A,D); 100 µm (B,C,E,F); 100 µm (G-J); 100 µm (K-N).
Fig. 2. MAP2-positive layers are formed by neurons which have been
dispersed by tangential migration. Electroporation was performed at E4.5 into
a portion of the tectal wall to transfect tol2 transposon-mediated expression
constructs for stable expression of EGFP. Transfected tectum at E8.5 was
sectioned perpendicular to the anterior-posterior axis of the tectum (dorsal is
left). (A-F) A section stained in combination with GFP, MAP2 and DAPI. Most of
the GFP-labeled cells were radially arranged in the transfected area (the area
denoted by E.P. in A). Two populations of the labeled cells emigrated
tangentially from the transfected area in ventral and dorsal directions (white and
yellow arrows, respectively, in A). Staining with an antibody against MAP2
revealed that the tangential migrants were distributed in MAP2-positive layers
(layers IV/V). (G-I) Staining with GFP (G,H) and MAP2 (H,I) at higher
magnification. GFP-labeled cells in layers IV/V are MAP2-positive, indicating
that the cells which migrated tangentially differentiated into MAP2-positive
neurons. Scale bars: 100 µm (A-F), 20 µm (G-I).
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DEVELOPMENT
It has been reported that some of the radially migrating cells in the
developing chick optic tectum change direction to the tangential
route and migrate to the intermediate layers (Gray and Sanes, 1991).
We questioned whether these layers correspond to the MAP2positive layers IV/V and examined the tangential migration in the
tectum. The tectal cells were traced by using stable labeling through a
transposon-mediated gene transfer system (Sato et al., 2007). The
expression vectors encoding tol2 transposase (pCAGGS-T2TP) and
enhanced green fluorescent protein (EGFP; pT2K-CAGGS-EGFP)
were co-electroporated into a small area of the ventricular layer of the
optic tectum at E4.5. At 4 days after electroporation (E8.5), in
sections perpendicular to the anterior-posterior axis of the tectum,
most of the GFP-labeled cells were radially arranged in the
transfected area (Fig. 2A). These cells might have been labeled in
the ventricular layer and radially migrated to the upper layers
(Fig. 2A,D). Some labeled cells were horizontally arranged in
layers IV/V. These cells might have emigrated tangentially from the
transfected area in ventral and dorsal directions (Fig. 2A). Staining
with an antibody against MAP2 revealed that the tangential migrants
were distributed in MAP2-positive neuronal layers (Fig. 2B,E). GFPlabeled cells in layers IV/V were large and multipolar, as well as
MAP2-positive (Fig. 2G-I), indicating that these cells correspond to
various transitional forms of ganglion cells or multipolar neurons
(Domesick and Morest, 1977; Puelles and Bendala, 1978; Gray and
Sanes, 1991).
We next examined the spatio-temporal sequence of the tangential
cell migration. At E6.5, 2 days after electroporation, a group of GFPlabeled cells, which elongated horizontally, migrated ventrally from
the transfected area into the intermediate zone (Fig. 3A,B). Cell
nuclei were labeled using co-electroporation of the mCherry gene
cassette that included a nuclear localization signal (mCherry-Nuc).
Serial distribution of the mCherry-positive nuclei indicated that the
tangential migration continued successively (Fig. 3C). The migrating
cells extended the tangential process toward the ventral direction,
parallel to the tectal efferent axons running dorsoventrally, which
expressed NgCAM (the chick homolog of the L1 cell adhesion
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
molecule), the marker of the tectal efferent axons (Fig. 3D; Kröger
and Schwarz, 1990). At E7.5, the labeled cells migrated more
ventrally and also dorsally in the intermediate zone (Fig. 3E). The
migrating cells exhibited a bipolar shape, typical to the directionally
migrating cells (Fig. 3H). At E8.5, the tangential migrants in the
ventral and dorsal directions traveled long distances in order to be
distributed throughout the whole tectum (Fig. 3F). Most of the
migrants were located in layers IV/V, indicating that they had left the
intermediate zone, which corresponded to layer III at E8.5, and
translocated to the upper layers in order to differentiate into various
forms of multipolar neurons (Fig. 3I). At E10.5, the tangential
migrants eventually formed arborized multipolar neurons in layers
IV/V (Fig. 3G,J). We noticed that the tangential migrants also existed
in the superficial layer after E7.5 (Fig. 3E-G; arrowheads), which
corresponded to the second tangential migration, which has been
reported previously (Gray and Sanes, 1991).
Movement of tangential cell migration along the tectal
efferent axons
Taking advantage of stable labeling, we observed the movement of
the tangential cell migration in a flat-mount culture. First, we
Fig. 3. Spatial and temporal distribution of the tangentially migrating cells. Electroporation was performed at E4.5 for stable expression of EGFP and mCherryNuc (A-D) or EGFP (E-J). Fluorescently labeled tectum at E6.5 (A-D), E7.5 (E,H), E8.5 (F,I) and E10.5 (G,J) were sectioned perpendicular to the anterior-posterior
axis of the tectum and immunostained. H-J are higher magnification images at E7.5, E8.5 and E10.5, respectively. (A) At E6.5, a group of GFP-labeled cells migrated
ventrally from the transfected area. The area enclosed by a white box is shown in higher magnification in B and C. (B) GFP-labeled cells, which were elongating
horizontally, migrated ventrally from the transfected area into the intermediate zone (IZ; white arrows). (C) mCherry-Nuc labeled nuclei of the tangentially migrating
cells. (D) The migrating cells (white arrow) had a leading process that elongated parallel to NgCAM-positive tectal efferent axons running dorsoventrally. (E,H) Cells
tangentially migrating in a dorsal direction were also observed in the intermediate zone at E7.5 (yellow arrows), in addition to the ventrally migrating cells (white
arrows). The migrating cells exhibited a bipolar shape. (F,I) At E8.5, the tangential migrants in the ventral and dorsal directions traveled a long distance to be
distributed throughout the whole tectum and translocated to the upper layers, IV/V (white and yellow arrows), as differentiating multipolar neurons. Another tangential
migration in the superficial layer was observed (white arrowheads). (G,J) Cells after tangential migration eventually formed arborized multipolar neurons in layers IV/V
(white and yellow arrows). E.P., dorsoventral area of electroporation. Scale bars: 100 µm (A-D); 1 mm (E-G), 100 µm (H-J).
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characterized the tectal efferent axons by examining their
destinations. When a portion of tectal wall was labeled at E4.5 by
using EGFP, the tectal efferent axons originating from the
transfected area were labeled with GFP at E6.5, which elongated
in parallel arrays to the ventral direction (Fig. 4A; n=3, three
embryos were examined). At E10.5, the labeled axons reached the
thalamic region ipsilaterally, and also contralaterally through the
ventral supraoptic decussation (Fig. 4B; n=5). They were eventually
destined for the rotundus nucleus bilaterally at E12.5 (Fig. 4C; n=3).
The projection of the tectal efferent axons indicated that the labeled
axons constitute the tectofugal pathway, and these axons had
tectorotundal projections (Deng and Rogers, 1998; Wu et al., 2000;
Puelles et al., 2007; Fedtsova et al., 2008). Next, we labeled tectal
cells with GFP and mCherry-Nuc to follow the tangentially
migrating cells. In the isolated tectum, GFP-labeled tectal efferent
axons elongated ventrally and turned anteriorly toward the
diencephalon at E7.0 (Fig. 4D). Within the ventral part of the
transfected area, many GFP-labeled cells with mCherry-positive
nuclei were observed, indicating that these cells had migrated
ventrally from the transfected area (Fig. 4E; n=3).
To monitor the dorsoventral movement of these tangentially
migrating cells in a flat-mount culture, three-dimensional images of
GFP, mCherry and differential interference contrast (DIC) at E6.0
were captured by using a confocal laser microscope (n=4). A number
of mCherry-Nuc-positive cells moved ventrally along the GFPlabeled axon fasciculi of the tectal efferents (supplementary material
Movie 1), which elongated dorsoventrally in the intermediate zone,
as shown in the sections in Fig. 3B-E. The moving nuclei typically
exhibited an oblong shape along the proceeding direction
(supplementary material Movie 1, mCherry-Nuc). Moreover,
mCherry-Nuc-positive cell nuclei proceeded in contact with the
GFP-labeled tectal axon fasciculi, which were recognized in white
arrays in DIC (supplementary material Movie 1, lower panels). Upon
imaging at a higher magnification (supplementary material Movie 2;
Fig. 5A-I), we noticed that the migrating cells held a single motile
leading process, which traced pre-existing GFP-positive tectal axons
(supplementary material Movie 2, left panels). Moreover, the nuclei
proceeded on the tectal axons or moved adjacently to them
(supplementary material Movie 2, lower panels; Fig. 5A-C,G-I).
The clinging movements along the tectal axons were most evident on
the tracking images of the frames (Fig. 5J-L). Trajectories of the cell
(GFP) and the nuclei (mCherry) stayed adhered along the
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
dorsoventral array of the tectal axon fasciculi (DIC), suggesting
that the cells migrate on the passages of tectal axons. We further
confirmed the attachment of migrating cells on tectal axons in fixed
specimens. In confocal images, GFP-labeled migrating cells with
mCherry-labeled nuclei were tightly attached to the bundles of tectal
axons, which were also labeled by GFP (Fig. 5M). Immunostaining
of NgCAM, the marker for tectal axons (Kroger and Schwarz, 1990),
revealed that the migrating cell, the shape of which was marked with
GFP, was attached to NgCAM-positive bundles, meaning that
migrating cells proceeded in contact with the tectal axon fasciculus
(Fig. 5N). We noticed that most of the GFP-positive bipolar cells had
not only a leading process but also a very thin trailing process, which
elongated from the soma (Fig. 5N). In the area just dorsal to the site
of transfection, we found that the cells tangentially migrating in the
ventral to dorsal direction also held a leading and a trailing process
and attached to NgCAM-positive tectal axons (Fig. 5O,P). Finally,
axophilic movement of tangentially migrating cells was confirmed
by removing the axons on which the cells proceeded. After cutting
the ventral tectal tissue, including the distal end of the tectal efferent
axons, we observed the behavior of dorsoventral cell migration
(supplementary material Movie 3). Focusing on the movement of
mCherry-Nuc-positive cell nuclei, the migrating cells stopped at the
distal tip of the axons and then turned back in the opposite direction
(arrowheads) or restarted, proceeding in the same direction as the
following axons elongated (arrows). In both cases, the migrating
cells persistently traced the axonal bundles. These features of cell
migration imply that the tangentially migrating cells move ventrally
and dorsally along the tectal efferent axons.
Mis-expression of Sema3A disrupts tectal laminar
organization
The results so far indicate that the tectal intermediate layers (layers
IV/V) are formed by the cells that have migrated tangentially and
differentiated into MAP2-positive neurons. Because NRP1 is
expressed in layers IV/V, and Sema3A is expressed in the overlying
layer VI, we sought to elucidate the roles of NRP1 and Sema3A in the
tectal laminar formation. To this end, Sema3A was mis-expressed
with GFP into the tectum by using the Tet-On system to limit the time
window of the mis-expression after E6.5 when the lamination of the
tectum proceeds rapidly (Fig. 6A,B; Sato et al., 2007). Induction of
Sema3A-mis-expression had already started 10 h after doxycycline
injection (Fig. 6C), and mis-expressed Sema3A was detected in
Fig. 4. Route of tangential migration along the
tectal efferent axons. Optic tectum was labeled with
GFP (A-C) or GFP and mCherry-Nuc (D,E) by
electroporation at E4.5. (A) In isolated whole tectum at
E6.5, GFP-labeled efferent axons that had originated
from the dorsal transfected area elongated ventrally.
(B) In the ventral view of the whole brain at E10.5,
GFP-positive axons reached the thalamic area
ipsilaterally, and contralaterally through the ventral
supraoptic decussation (xsov). (C) In the transverse
section at E12.5, the labeled axons bilaterally
projected toward the rotundus nucleus (Rt), enclosed
in white dotted circles. (D) In the whole tectum at E7.0,
GFP labeled two distinct areas of the tectum and
tracts of the tectal efferent axons projecting from them.
The area enclosed by the white box is shown in higher
magnification in E. (E) Within the ventral part of the
transfected area, many GFP-labeled cells with
mCherry-positive nuclei were observed, which
migrated ventrally. a, anterior; p, posterior; d, dorsal; v,
ventral; R, right; L, left; ipsi, ipsilateral; contra,
contralateral. Scale bars: 1 mm (A-D), 100 µm (E).
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Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
Fig. 5. Tangential migration along the tectal efferent axons. Movement of the tangentially migrating cells along the tectal efferent axons was shown in flatmount culture (A-L) and in fixed specimens (M-P). Images at 0, 12 and 24 h of culture time (A-I), and the tracking images of the frames over 24 h (J-L) are shown.
Note that trajectories of the migrating cells (GFP) and their nuclei (mCherry) were attached to the dorsoventral array of the tectal axon fasciculi (DIC). A time-lapse
movie is shown in supplementary material Movie 2. In confocal images of fixed specimens, bipolar-shaped GFP-labeled migrating cells with mCherry-labeled
nuclei were tightly attached to the bundles of the tectal efferent axons (M), which were marked by using NgCAM staining (N; higher magnification). In the area just
dorsal to the transfected site, GFP-labeled ventro-dorsally migrating cells were also attached to NgCAM-positive tectal axons (O,P; higher magnification). Dorsal
is toward the top of the image. Scale bars: 100 µm (A-M,O); 10 µm (N,P).
Mis-expression of Sema3A eliminates MAP2-positive
neurons that express NRP1
To explore the reasons for the disorganization of the tectal laminae
after Sema3A mis-expression, we focused on the MAP2-positive
neurons that expressed NRP1. At E8.5, two days after induction of
Sema3A expression, MAP2-positive neurons in layers IV/V were
completely lacking around the radial columns where Sema3A was
mis-expressed (Fig. 7A, arrows; n=6). The undulation of layer VI
was not evident at this time point (Fig. 7A, arrowheads). At E9.5,
three days after induction, undulation of layer VI became prominent
(Fig. 7B, arrowheads) at the region where the underlying MAP2positive neurons were lacking (Fig. 7B, arrows; n=3). This time
sequence suggests that Sema3A first eliminated MAP2-positive
neurons and subsequently yielded undulation of layer VI. When
Sema3A was induced much later at E8.5, the mis-expression area
was located in upper layers, such as layers VI, VII and VIII
(Fig. 7C), and MAP2-positive neurons were partly eliminated
(Fig. 7C, arrows; n=3). However, the upper layers VI-VIII were not
severely affected (Fig. 7C, arrowheads). These results suggest that
disruption of the tectal layers occurred after the elimination of the
underlying layers. It was confirmed that NRP1-expressing neurons
in layers IV/V were eliminated (Fig. 7D, arrows) around the region
where Sema3A was mis-expressed (Fig. 7D, arrowheads; n=6),
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whereas NgCAM-positive efferent axons in layer III were
unaffected (Fig. 7E; n=6). Mis-expression of GFP in the control
did not change the distribution of MAP2- or NRP1-expressing
neurons (Fig. 7F,G; n=7).
Sema3A repels tangential migrants that form multipolar
neurons
Because MAP2- and NRP1-positive neurons after tangential
migration were eliminated by the mis-expression of Sema3A, we
investigated whether excess of Sema3A might have repelled the
tangential migrants during their disposition to layers IV/V. To verify
this, the tangentially migrating cells were pre-labeled with mCherry
by electroporation at E4.5, and later Sema3A expression was induced
at E6.5, as in the previous experiment. At E8.5, mCherry-labeled cells
were radially arranged in the area of electroporation (Fig. 8A), and
some of them migrated tangentially and localized in layers IV/V
(Fig. 8A). However, mCherry-labeled tangential migrants were not
arranged continuously but were partially lacking around the sites of
Sema3A induction where GFP was expressed (Fig. 8A,B; n=7). The
density of the mCherry-labeled cells was high next to the Sema3A
induction site (Fig. 8A). In some cases, mCherry-labeled cells were
eliminated from the area where Sema3A was induced and were mislocated, away from the induction site (Fig. 8C-E). In the control
experiments, GFP mis-expression did not disturb the distribution of
the mCherry-labeled cells in layers IV/V (Fig. 8F-H; n=7). These
defects of cell disposition imply that Sema3A diffuses from the
source of its mis-expression to repel tangential migrants and prevent
them from forming layers IV/V.
To demonstrate the repulsive activity of Sema3A on the tangential
migrants, we monitored the behavior of the mCherry-labeled cells
during the induction of Sema3A in tectal slice culture. The tectal slices
at E8.5 containing mCherry-labeled multipolar neurons after
DEVELOPMENT
radially arranged cells covering the tectal layers at E8.5 (Fig. 6D). At
E10.5, tectal laminae were severely undulated; in particular, layer VI
expanded upward (Fig. 6E), where radially arranged columns of
GFP-positive cells were located (Fig. 6F; n=11, 11 embryos were
examined). In the control, transfection of EGFP alone did not affect
the laminar organization (Fig. 6G, H; n=4). Caspase 3 staining
indicated that apoptotic cell death was not induced by mis-expression
of Sema3A (Fig. 6I, J; n=4).
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Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
By contrast, the multipolar neurons outside of the induction site
(arrows) remained in their original positions. The induction of GFP
alone did not evoke the movement of the multipolar neurons
(supplementary material Movie 5; n=3). These observations
confirm that Sema3A repels tangential migrants that form
multipolar neurons.
Fig. 6. Mis-expression of Sema3A disrupts tectal laminar organization.
(A) Schematic representation for electroporation of the expression constructs
at E1.5 into the prospective optic tectum. (B) Overview of the procedure for
Sema3A induction through doxycycline (Dox) injection. (C,D) In situ
hybridization pattern showing the expression of Sema3A 10 h (C) and 48 h (D)
after doxycycline injection. (E,F) Tectal layers at E10.5 after induction of
Sema3A and EGFP at E6.5. Mis-expression of Sema3A from E6.5 to E10.5
disrupted the tectal layers, especially on layer VI. Note that upper undulation of
layer VI coincides with the radial localization of GFP, where exogenous
Sema3A expression was induced. (G,H) Induction of EGFP alone had no
effect on the laminar formation. (I,J) Staining with an antibody against Caspase
3 (Cas3) at E8.5 revealed that apoptotic cell death was not induced by Sema3A
mis-expression. Scale bars: 100 µm (C-J).
tangential migration were cultured in doxycycline-containing medium
to induce Sema3A and GFP expression. The multipolar neurons
initially residing within the transfected radial column (arrowheads)
migrated out from the area of increasing GFP signal (white box) where
Sema3A was mis-expressed (supplementary material Movie 4; n=3).
Mis-expression of Sema3A eliminated MAP2- and NRP1-positive
neurons in layers IV/V and resulted in the disorganization of tectal
layers, such as layer VI. We suspected that the radial migration of
the tectal cells to the upper layers might have been affected after
Sema3A mis-expression, which resulted in the formation of
undulated layers. After induction of the transgenes at E6.5, we
injected 5-bromo-2′-deoxyuridine (BrdU) into the aqueduct of the
tectal hemispheres at E7.0 and visualized BrdU-labeled cells 48 h
later at E9.0. The radial distribution of BrdU-labeled cells that had
migrated from the ventricular layer was compared between the
radial columns of the transfected tectal hemisphere and the control
untransfected hemisphere (Fig. 9A,B and Fig. 9C,D, respectively).
In the control sections, most of the BrdU-labeled cells were
scattered in and under layer VI but scarcely reached over layer VI
(Fig. 9C,D). In the experimental sections, the labeled cells were
distributed in all layers, and some of them reached the superficial
layers (Fig. 9A,B). To check the different distribution of the BrdUlabeled cells along the radial axis, the cumulative proportion of
each BrdU-labeled cell was plotted against a relative migration
distance from the ventricular zone to the pial surface (Fig. 9E). We
noticed that the plots of the experimental section (red plots; n=69)
had a longer migration distance than that of the control section
(blue plots; n=68). To quantify the overall distribution of the
BrdU-labeled cells, we divided the tectal layers outside the
ventricular zone into ten equally spaced bins along the radial axis
(Fig. 9B,D) and obtained the proportion of the BrdU-labeled cells
within each bin (Fig. 9F; each of the ten sections from five embryos
that had been transfected with Sema3A plus EGFP). A line chart of
the control sections (blue line) peaked in the middle layers (bins 5
and 6) and declined rapidly in the upper layers (bins 7-9), whereas
that of the experimental sections (red line) sloped gently in the
middle to the upper layers (bins 4-9), suggesting a shift of cell
distribution toward the upper layers. Finally, we compared the
cumulative proportion of the BrdU-labeled cells along the radial
axis (Fig. 9G). The accumulation of the BrdU-labeled cells toward
the upper layers was similar between the experimental and control
sections in EGFP-transfected embryos (green and orange line,
respectively; each of the ten sections from four EGFP-transfected
embryos). By contrast, in embryos that had been transfected with
Sema3A plus EGFP, the accumulation curve of the experimental
sections (red line) was shifted distinctively toward the upper layers
from that of the control sections (blue line). These analyses
confirm that radial migration to the upper layers is enhanced after
Sema3A mis-expression.
DISCUSSION
In the present study, we obtained the following results: (1) Sema3A
and NRP1 are expressed complementarily, Sema3A in layer VI
and NRP1 in layers IV/V; (2) MAP2-positive layers IV/V are
formed from neurons that have migrated tangentially along the tectal
efferent axons; (3) mis-expression of Sema3A eliminates MAP2positive neurons that express NRP1 and results in undulated laminar
organization; (4) Sema3A repels the tangential migrants; and (5)
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DEVELOPMENT
Radial migration to the upper layers is enhanced after
mis-expression of Sema3A
RESEARCH ARTICLE
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
radial migration to the upper layers is enhanced after mis-expression
of Sema3A. Based on these results, a possible role of NRP1mediated Sema3A signals in laminar formation of the developing
optic tectum is indicated.
Tracing of the stably labeled cells during tectal layer formation
revealed that some of the radially migrating cells change direction to
the tangential route and span a very long distance. Time-lapse
imaging analysis demonstrated that the tangentially migrating cells
trace the bundles of pre-existing tectal efferent axons, which serve as
the substrate for migration. These results confirm a previous report
that associates tangential migration with axon fasciculus (Gray and
Sanes, 1991). An axonal substrate for tangential migration might
facilitate a successive migration stream over a long distance. It was
also shown that the tangential migrants differentiate into MAP2positive neurons in layers IV/V. Because MAP2-positive neurons
were distributed over the whole tectum, tangential migration is a
crucial process for the formation of layers IV/V.
We showed that Sema3A and NRP1 were expressed in the
complementary tectal layers. Class 3 semaphorins can function as
3578
guidance molecules, either as an attractant or repellent, depending
on the signal-receiving cells that express the receptors. The
mis-expression experiment of Sema3A revealed that MAP2positive neurons, which express NRP1, were eliminated around
the site of the mis-expressed Sema3A. This result indicates that
Sema3A in layer VI exerts repulsive effects on the NRP1-expressing
neurons in layers IV/V. Moreover, we found that multipolar
neurons, which were labeled by mCherry and displaced by
tangential migration, were repelled by mis-expressed Sema3A.
This suggests that mCherry-labeled cells after tangential migration
differentiate into multipolar neurons that express NRP1, which in
our study were repelled by locally induced Sema3A. Because the
density of the mCherry-labeled cells next to the Sema3A induction
site was high, repelled neurons might be excluded horizontally from
the Sema3A source as shown in the repulsion analysis using tectal
slice culture (supplementary material Movie 4).
In the developing tectum, Sema3A is expressed in layer VI cells,
which overlie layers IV/V cells, which express NRP1 (a schematic
model is shown in Fig. 10). During tectal layer formation, layer VI
DEVELOPMENT
Fig. 7. Sema3A eliminates MAP2-positive neurons that
express NRP1. The effects of Sema3A mis-expression on
the tectal layers were examined after different sequential
conditions of Sema3A mis-expression. (A) Tectal layers
after mis-expression of Sema3A during E6.5-E8.5. After
two days of Sema3A induction, MAP2-positive layers were
eliminated around the radial columns where Sema3A and
EGFP were mis-expressed (arrows), whereas layer VI was
affected only slightly (arrowheads). (B) Tectal layers after
Sema3A mis-expression during E6.5-E9.5. After three
days of induction, layer VI became undulated (arrowheads)
where underlying MAP2-positive layers were dismissed by
Sema3A mis-expression (arrows). (C) Tectal layers after
Sema3A mis-expression during E8.5-E10.5. Although
Sema3A was mis-expressed in upper layers, such as
layers VI, VII and VIII (arrowheads), these upper layers
were not severely affected. By contrast, MAP2-positive
layers were partly missing (arrows). (D,E) Tectal layers
after mis-expression of Sema3A during E6.5-E8.5.
NRP1-positive neurons were eliminated (D, arrows) around
the radial columns where Sema3A and EGFP were
mis-expressed (D, arrowheads). NgCAM-positive efferent
axons in layer III (E) were unaffected. (F,G) Mis-expression
of GFP alone did not change the distribution of MAP2- or
NRP1-expressing neurons. Scale bar: 100 µm.
RESEARCH ARTICLE
cells and layers IV/V cells have distinct birthdates and origins.
Among the three waves of radial migration, the first migration
between E3 and E5 forms the inner laminae, the second migration
between E4 and E7 forms the outer laminae and the third migration
between E5 and E8 forms the middle laminae (La Vail and Cowan,
1971b; Sugiyama and Nakamura, 2003). Cells of layers IV/V,
which are in the inner laminae, derive from radially migrating cells
during the first migration. After E5, these radially migrating cells
change direction and migrate tangentially along the tectal efferent
axons in the intermediate zone, which later corresponds to layer III.
During E7.5 to E8.5, the tangentially migrating cells translocate to
the upper layers, IV/V, to differentiate into NRP1-expressing
multipolar neurons. By contrast, layer VI cells, which are in the
middle laminae, are formed by the third radial migration after E5.
These radially migrating cells pass through layers III-V, while first
migrants move tangentially, and eventually form layer VI,
expressing Sema3A, which overlies differentiating multipolar
neurons after tangential migration. Given the repulsive activity of
Sema3A on the NRP1-expressing neurons, we suspect that
endogenous Sema3A in layer VI might act in the sorting of
NRP1-expressing neurons, that is to sort tangential migrants out of
Sema3A-expressing layer VI. We propose that Sema3A prevents
NRP1-expressing neurons in layers IV/V from entering layer VI in
order to segregate these layers. Because other class 3 semaphorins
are also expressed in layer VI (Y. W., unpublished), multiple
inhibitory semaphorin signals might redundantly protect the
segregation of the different layers.
The experiment in which Sema3A was mis-expressed revealed
that the disorganization of layer VI occurred after the elimination of
the underlying layers IV/V. Layer VI mainly comprises cells which
became post-mitotic in the ventricular zone and then migrate
radially between E5-E8 (La Vail and Cowan, 1971b; Sugiyama and
Nakamura, 2003). Because undulation of layer VI was not prominent
at E8.5, two days after the mis-expression of Sema3A, the absence of
layers IV/V did not disturb the initial formation of layer VI.
Thereafter, the thickness of layer VI became varied and deformed
into undulation at E9.5, suggesting that the loss of layers IV/V might
have triggered abnormal disposition of layer VI cells. In the cerebral
and cerebellar cortices, the undulation of the layer structure can be
caused by over-migration of the radially migrating cells (Moers et al.,
2008). Similarly, in tectal layer formation after Sema3A misexpression, proliferative tectal cells labeled with BrdU in the
ventricular zone migrated radially and were distributed into the more
upper layers than those of the control, suggesting that enhanced
radial migration can drive layer VI undulation. We propose that the
loss of NRP1-expressing cells upon Sema3A overexpression might
perforate layers IV/V, which might lead to further radial migration.
Supporting this idea, the loss of the tectal pial cells, which overlay
tectal layers, causes over-migration of the radially migrating cells,
therefore inducing undulation of the layers (McGowan et al., 2012).
These results indicate that the uniform distribution of layer IV/V
cells, which is elaborated by tangential migration, is required to
maintain lamination of layer VI cells after radial migration.
In summary, we propose a model of tectal laminar formation in
the developing chick optic tectum (Fig. 10). During laminar
formation, radial migration of post-mitotic cells from the ventricular
zone to the upper layers occurs in every area of the optic tectum.
Some of these cells take the tangential route in the intermediate
zone, which later corresponds to layer III, and proceed on the
fasciculus of the tectal efferent axons running along the dorsoventral
axis. Following broad dispersal over the whole tectal area, the cells,
after tangential migration, translocate to layers IV/V and
differentiate into MAP2-positive multipolar neurons, which
express NRP1. Sema3A is expressed in layer VI, which is formed
upon radial migration. Sema3A provides repulsive signals to
MAP2-positive neurons that express NRP1 and prevents them
from entering the upper layers. MAP2-positive multipolar neurons
in layers IV/V are required for proper laminar formation. Taken
together, the data of this study indicates a role for NRP1-mediated
Sema3A signals in the formation of the intermediate layers, which is
important to coordinate laminar organization of the optic tectum.
MATERIALS AND METHODS
Constructs
pCAGGS-T2TP (Tol2 transposase), pT2K-CAGGS-EGFP (stable EGFP
vector), pT2K-CAGGS-rtTA2sM2 (Tet-On activator) and pT2K-BI-EGFP
(Tet-On EGFP vector) were kindly provided by Yoshiko Takahashi (Sato
et al., 2007). The mCherry gene cassette of pmCherry-C1 (Clontech) and the
nuclear localizing signal of pDsRed2-Nuc (BD Biosciences) were inserted
into pT2K-CAGGS to form pT2K-CAGGS-mCherryNuc. The mCherry
gene cassette of pmCherry-C1 was inserted into pT2K-CAGGS to form
pT2K-CAGGS-mCherry. The ECFP gene cassette of pECFP-N1 (Clontech)
was inserted into pT2K-CAGGS to form pT2K-CAGGS-ECFP. A cDNA
fragment of chick Sema3A was amplified from a mixture of E3 and E4.5 chick
brain mRNA using reverse transcription PCR. The full-length coding region
was inserted into pT2K-BI-EGFP (pT2K-BI-EGFP-Sema3A).
3579
DEVELOPMENT
Fig. 8. Sema3A repels tangential migrants that form multipolar neurons.
The effects of Sema3A mis-expression on the tangential migrants were
examined. The tangentially migrating cells were pre-labeled by the
electroporation of an mCherry construct at E4.5, and Sema3A was misexpressed during E6.5-E8.5. (A,B) mCherry-labeled multipolar neurons, which
migrated tangentially from the transfected area (denoted with E.P.), were
horizontally aligned in layers IV/V. However, they were lacking locally around
the sites of Sema3A mis-expression where GFP was expressed (asterisks).
The density of the mCherry-labeled cells was high next to the Sema3A
induction site (arrows in A). (C-E) In some cases, mCherry-labeled cells were
eliminated from the area where Sema3A was induced (asterisk) and were mislocated, away from the induction site (arrow). (F-H) Mis-expression of GFP
alone did not disturb the distribution of mCherry-labeled cells in layers
IV/V. Staining in combination with mCherry (magenta; A-H), GFP (green;
B,D,E,G,H) and DAPI (cyan; E,H) are shown. Scale bars: 100 µm.
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
RESEARCH ARTICLE
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
Fig. 9. Enhanced radial migration after Sema3A mis-expression. (A-D) Radial migration of tectal cells labeled with BrdU after Sema3A mis-expression. After
induction of Sema3A at E6.5, BrdU was injected into the tectal hemispheres at E7.0, and BrdU-labeled cells were visualized 48 h later. Sections from the
hemisphere that had been transfected with Sema3A plus EGFP (A,B) were compared with those from a control untransfected hemisphere of the equivalent region
(C,D) Scale bar: 100 μm. Layers above layer VI of the normal embryos are depicted by a square bracket (B,D). (E) Distribution of BrdU-labeled cells in the radial
axis. Cumulative relative frequency of each labeled cell was plotted against a relative migration distance from the ventricular zone to the pial surface. n=69
cells (Experimental, Exp), n=68 cells (Control, Cont). (F,G) Quantitative analysis of the distribution of BrdU-labeled cells. The tectal layers outer to the ventricular
zone were divided into ten equally spaced bins along the radial axis, and the proportion of BrdU-labeled cells in each bin was obtained and averaged across
ten sections from five embryos (those transfected with Sema3A plus EGFP shown in F,G) or four embryos (EGFP-transfected shown in G). The relative frequency
(F) or cumulative relative frequency (G) of the labeled cells in each bin was plotted. Error bars represent the s.e.m.
Ex ovo culture of chick embryos was performed as previously described
(Nakamura and O’Leary, 1989; Luo and Redies, 2005). The contents of
fertile chicken eggs at E2.5 were transferred into a plastic petri dish.
At E4.5, a DNA solution of a mixture of pCAGGS-T2TP (2 µg/µl)
with pT2K-CAGGS-EGFP (5 µg/µl) or with pT2K-CAGGS-mCherry
(5 µg/µl), or a mixture of pCAGGS-T2TP (1 µg/µl) with pT2KCAGGS-EGFP (2 µg/µl) and pT2K-CAGGS-mCherryNuc (2 µg/µl)
was injected into the aqueduct of the left optic tectum with a
micropipette. A pair of forceps-type electrodes (LF646P3x3, Unique
Medical Imada, Japan) was set so that the target area of the optic tectum
was placed in between. Ex ovo electroporation was performed using a
pre-pulse of 30 V with a pulse length of 1 ms and a pulse interval of
5 ms, which was then proceeded by four pulses of 6 V with a pulse
length of 5 ms and a pulse interval of 10 ms, using a CUY21EX pulse
generator (BEX, Japan).
Fig. 10. A model for tectal laminar formation. In the tectal
development, bipolar-shaped cells (red) migrate tangentially (red
arrows) on the axon fasciculus of the tectal efferents (gray), which run
along the dorsoventral axis in layer III, in order to broadly disperse over
the whole tectal area. Cells after tangential migration differentiate into
MAP2-positive multipolar neurons (magenta) in layers IV/V, which
express NRP1. Sema3A (blue) is expressed in layer VI, which is
formed by radial migration (cyan). Sema3A provides repulsive signals
to MAP2-positive neurons that express NRP1 and segregates them
from entering the upper layers.
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DEVELOPMENT
Ex ovo and in ovo electroporation
RESEARCH ARTICLE
In ovo electroporation was performed at E1.5 (stage 10-11, Hamburger
and Hamilton, 1951) as described previously (Funahashi et al., 1999;
Watanabe and Nakamura, 2000). For Tet-On activation of Sema3A by
doxycycline, pT2K-BI-EGFP-Sema3A (3 µg/µl) or control pT2K-BI-EGFP
(3 µg/µl) was mixed with pCAGGS-T2TP (1 µg/µl) and pT2K-CAGGSrtTA2sM2 (1 µg/µl) and injected into the mesencephalon, between a pair of
electrodes (LF610P4x1, Unique Medical Imada). Four pulses of 25 V,
50 ms were charged in a one-second interval by the electroporator
(CUY21EDIT, BEX). At 5 and/or 7 days after the electroporation (E6.5,
E8.5), 100 µl of doxycycline (0.2 mg/ml) was injected into the yolk with a
micropipette to induce transgene expression.
Flat-mount culture
The optic tectum from the embryo that had been electroporated ex ovo at
E4.5 was dissected out at E6.0 in ice-cold Hanks’ balanced salt solution
(HBSS). The tectum was cut transversely with a micro scalpel and laid in a
flat-mount on a Millicell CM-ORG cell culture insert (Millipore), which
was set on a 35-mm glass-bottomed dish (Matsunami) filled with culture
medium (60% Opti-MEM, 20% F12, 10% fetal bovine serum, 10% chick
serum, 50 units/ml penicillin, 50 μg/ml streptomycin). The dish was then
incubated on an inverted microscope (IX81, Olympus, Japan) in a humid
chamber unit with a gas flow of 40% O2 and 5% CO2 and a controlled
temperature of 38°C. Images of GFP and mCherry fluorescence with DIC
were captured by using a confocal laser scanning microscope (FV300,
Olympus) every 5 µm along the z-axis to a depth of 60-80 µm. z-stack
images at every 10 min interval during a period of 18-24 h were collected to
construct a time-lapse movie.
Development (2014) 141, 3572-3582 doi:10.1242/dev.110205
Repulsion analysis in slice culture
In ovo electroporation of pT2K-BI-EGFP-Sema3A (3 µg/µl) or control
pT2K-BI-EGFP (3 µg/µl) with pCAGGS-T2TP (1 µg/µl), pT2K-CAGGSrtTA2sM2 (1 µg/µl) and pT2K-CAGGS-ECFP (1 µg/µl) was applied to the
left mesencephalon at E1.5. At E4.5, pCAGGS-T2TP (1 µg/µl) with pT2KCAGGS-mCherry (5 µg/µl) was electroporated into the left optic tectum. At
E8.5, the optic tectum that had been labeled with cyan fluorescent protein
(CFP) and mCherry was embedded with 3% low melting agarose and sliced
at 250 µm with the vibratome. The tectal slices were mounted in collagen gel
(60% rat tail collagen in Dulbecco’s modified Eagle medium) in a glassbottomed dish (Matsunami) and covered with 1 ml of the culture medium
(described in flat-mount culture) supplemented with doxycycline (2 µg/ml).
Prospective GFP-positive areas after doxycycline-mediated induction
were identified with the co-electroporated CFP signal. mCherry-labeled
multipolar cells in CFP-positive radial columns were focused under a
confocal laser microscope in the culture chamber system described above.
The behavior of mCherry-labeled cells during the induction of Sema3A and
GFP or control GFP was monitored every 10 min for 20 h.
Acknowledgements
We thank Yoshiko Takahashi and Koichi Kawakami for providing the tol2-mediated
transgene vectors, and Masahito Yamagata for the chick NRP1 antibody (TB2;
available in DSHB). We also thank Harukazu Nakamura for his critical reading of the
manuscript. The 8D9 monoclonal antibody was obtained from the DSHB developed
under the auspices of the National Institute of Child Health and Human Development
and maintained by The University of Iowa, Department of Biology, Iowa City,
IA52242.
Competing interests
Immunostaining
Cryosections were stained with the following monoclonal antibodies: MA512826 (anti-MAP2, Thermo Scientific), 8D9 [anti-NgCAM, Developmental
Studies Hybridoma Bank (DSHB)], TB2 (anti-NRP1, kindly provided by
Masahito Yamagata; Yamagata et al., 2006) or an anti-red fluorescent protein
(PM005, MBL) polyclonal antibody. AlexaFluor594-labeled anti-mouse or
anti-rabbit IgG secondary antibodies were used (Invitrogen). The GFP signal
was enhanced using an anti-GFP polyclonal (A6455, Invitrogen) or an
anti-GFP monoclonal (012-20461, Wako) antibody with an Alexa488labeled secondary antibody (Invitrogen). Fluorescence images were captured
by using a cooled charge-coupled device digital camera (ORCA-ER,
Hamamatsu, Japan) or a confocal laser scanning microscope (FV300,
Olympus).
The authors declare no competing financial interests.
Author contributions
Y.W. designed the study, analyzed the data and prepared the manuscript. Y.W. and
C.S. performed the experiments. H.Y. developed the approach and edited the
manuscript.
Funding
This study was partly supported by Fukushima Medical University Research Project.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.110205/-/DC1
In situ hybridization of cryosections was performed as previously described
(Henrique, 1997; Watanabe and Nakamura, 2000). Antisense digoxigeninlabeled RNA probes were used for the coding region and the 3′-UTR of the
chick Sema3A mRNA (3 kb downstream from nucleotide 2431 of the
coding region) or for the coding region only (1.1 kb).
AP-fusion protein binding assay
Chick NRP1 ectodomain fused with alkaline phosphatase at its C-terminal
(NRP1-AP) was prepared as previously described (Watanabe et al., 2004).
Binding of NRP1-AP to the ligands was confirmed in COS7 cells that had
been transfected with expression vectors containing chick Sema3A cDNA
(supplementary material Fig. S1).
BrdU-labeling and migration analysis
Fifteen microliters of 5-bromo-2′-deoxyuridine (BrdU; 1 mg/ml) was
injected into the aqueduct of the tectal hemispheres at E7.0. The tectal
hemispheres were fixed 48 h later at E9.0. Cryosections were pre-treated
with 2.0 M hydrochloric acid (HCl) at 37°C for 20 min and then neutralized
with 0.1 M boric acid ( pH 8.5) at room temperature for 30 min to detect
BrdU-labeled cells with a monoclonal antibody (010198, BioScience
Products), and nuclei were stained using DAPI. Distance from the
ventricular zone to the BrdU-labeled cells was measured within the
transfected radial column with a width of 100 µm, using ImageJ software to
analyze the radial migration after mis-expression.
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