Position and time specify the migration of a pioneering population of
olfactory bulb interneurons
Eric S. Tuckera, Franck Polleuxb, Anthony-Samuel LaMantiaa,⁎
aDepartment of Cell and Molecular Physiology, UNC Neuroscience Center, The University of North Carolina at Chapel Hill School of Medicine,
Chapel Hill, NC 27599, USA
bDepartment of Pharmacology, UNC Neuroscience Center, The University of North Carolina at Chapel Hill School of Medicine,
Chapel Hill, NC 27599, USA
Received for publication 20 March 2006; revised 29 April 2006; accepted 5 May 2006
Available online 19 May 2006
We defined the cellular mechanisms for genesis, migration, and differentiation of the initial population of olfactory bulb (OB) interneurons.
This cohort of early generated cells, many of which become postmitotic on embryonic day (E) 14.5, differentiates into a wide range of mature OB
interneurons by postnatal day (P) 21, and a substantial number remains in the OB at P60. Their precursors autonomously acquire a distinct identity
defined by their position in the lateral ganglionic eminence (LGE). The progeny migrate selectively to the OB rudiment in a pathway that presages
the rostral migratory stream. After arriving in the OB rudiment, these early generated cells acquire cellular and molecular hallmarks of OB
interneurons. Other precursors – including those from the medial ganglionic eminence (MGE) and OB – fail to generate neuroblasts with similar
migratory capacity when transplanted to the LGE. The positional identity and migratory specificity of the LGE precursors is rigidly established
between E12.5 and E14.5. Thus, the pioneering population of OB interneurons is generated from spatially and temporally determined LGE
precursors whose progeny uniquely recognize a distinct migratory trajectory.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Migration; Interneuron; Olfactory bulb; Development; Forebrain; Neurogenesis; Ganglionic eminence; Rostral migratory stream
The production, migration, and differentiation of the earliest
generated OB interneurons have largely been unexplored,
despite considerable attention to the genesis of these cells
during early postnatal life and adulthood (Hinds, 1968a;
Rosselli-Austin and Altman, 1979; Bayer, 1983; Lois and
Alvarez-Buylla, 1993; Luskin, 1993; Lois and Alvarez-Buylla,
1994; Alvarez-Buylla and Lim, 2004). Genetic loss-of-function
and fate mapping studies (Corbin et al., 2000; Wichterle et al.,
2001; Yun et al., 2001, 2003) clearly demonstrate that
precursors in the LGE produce progeny that migrate into the
rudimentary OB and differentiate into OB interneurons. The
cellular and molecular specificity of this population, however,
has not been defined. Moreover,it is unclear whether the earliest
cohort of cells from the LGE migrate along a specific pathway
to the OB rudiment and differentiate into a full range of OB
interneurons that persist throughout life, perhaps as a scaffold
upon which the adult compliment is established postnatally and
replaced thereafter. Finally, it is not known when the production
of embryonic OB interneurons becomes restricted to the LGE
during development. Accordingly, we used in vivo neuronal
birth dating, molecular labeling, and a novel in vitro assay to
determine the identity of embryonically generated OB inter-
neurons and their precursors, and define their migratory route to
the rudimentary OB.
Concurrent with the production of presumptive OB inter-
neurons in the LGE, precursors in the adjacent MGE generate
cortical, striatal, and hippocampal interneurons (Anderson et al.,
1997; Marin et al., 2000; Pleasure et al., 2000; Anderson et al.,
2001; Wichterle et al., 2001). The extent to which position in the
ganglionic eminences controls specific aspects of neuronal
identity, such as the selection of migratory pathways and
differentiation programs, remains unclear. Previous reports
Developmental Biology 297 (2006) 387–401
⁎Corresponding author. Fax: +1 919 966 6927.
E-mail address: firstname.lastname@example.org (A.-S. LaMantia).
0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
indicate that precursors from the LGE, MGE, and caudal
ganglionic eminence (CGE) display a significant degree of
intrinsic determination (Wichterle et al., 1999; Wichterle et al.,
2001; Nery et al., 2002; Yozu et al., 2005); nevertheless, the
immediate influence of extrinsic factors available in each
location is unknown. We therefore used our novel in vitro whole
telencephalon assay, which preserves the in vivo architecture of
the developing forebrain, to ask whether autonomous factors or
local signals within the LGE distinguish OB interneuron
precursors and influence their subsequent migration and
We found that OB interneuron precursors in the LGE are
positionally specified and autonomously acquire a distinct
migratory capacity between E12.5 and E14.5. These precursors
give rise to a heterogeneous population of stable OB
interneurons. Local LGE or MGE cues are insufficient to
reprogram the migratory fate of heterologous precursors;
nevertheless, position in the LGE is required for significant
migration of LGE cells into the OB rudiment. Apparently,
patterning mechanisms that establish distinct domains in the
ventral forebrain confer LGE precursors with the unique ability
to migrate to the OB and acquire distinctive characteristics
associated with OB interneurons.
Materials and methods
Wild-type Institute of Cancer Research(ICR) mice and mice hemizygous for
a Enhanced Yellow Fluorescent Protein (EYFP) gene under control of the
chicken beta actin promoter/cytomegalovirus immediate early enhancer [TgN
(ActbEYFP)1Nagy (Jax 003772)] were maintained by the University of North
Carolina at Chapel Hill Department of Laboratory Animal of Medicine. Wild-
type females were paired with hemizygous EYFP males to generate timed
pregnancies (day of vaginal plug = E0.5). Timed-pregnant mice were sacrificed
by rapid cervical dislocation, and embryos harvested for tissue culture or fixed
for immunocytochemical analysis. Adult animals used for immunocytochem-
istry were deeply anesthetized with urethane (2 mg/kg) and perfusion fixed. All
experimental procedures were reviewed and approved by the UNC-CH
Institutional Animal Care and Use Committee.
Timed-pregnant mothers were injected intraperitoneally on E14.5 with 5-
bromo-2-deoxyuridine (BrdU; Sigma, St. Louis, MO; dissolved in 0.9% NaCl
with 0.07 N NaOH) at 50 mg/kg followed by an intraperitoneal injection of
thymidine 1 h later (Sigma, St. Louis, MO; 500 mg/kg, dissolved in 0.9% NaCl
with 0.07 N NaOH) to limit availability of BrdU to mitotically active cells to a
brief interval. Litters were harvested 6-h post BrdU injection for the E14.5 time
point andeach subsequentday fromE15.5to postnatalday 0 (P0). Forlong-term
analyses, E14.5 timed-pregnant animals were injected intraperitoneally with a
single 50 mg/kg dose of BrdU, and mice from those litters were sacrificed on
P21 and P60.
Embryonic brains (up to E17.5) were dissected in Dulbecco's phosphate-
buffered saline (PBS; pH = 7.4) and immersion fixed overnight in 4%
paraformaldehyde inPBS (pH = 7.4).E18.5embryosandP0pupswere perfused
in the same fixative and brains postfixed 2 h at RT. P21 and P60 mice were
perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, 4% sucrose
(pH = 7.4). Fixed brains were rinsed through graded sucrose solutions (10%–
30%), embedded in agar, and frozen in cryo-embedding compound (OCT) using
liquid-nitrogen-cooled 2-methyl-butane. These blocks were stored at −80°C,
and 12-μm cryosections were cut at −20°C.
Sections were rehydrated in PBS, incubated in 2 N HCl for 30 min at 37°C,
rinsed 3 × 15 min in a 3.8% sodium borate solution (pH = 8.5), 2 × 10 min in
PBS and then incubated for 1.5 h in 3% bovine serum albumin (BSA), 10%
normal goat serum (NGS), 0.3% Triton X-100, and 0.1% sodium azide. Mouse
anti-BrdU (1:100; Beckton Dickinson) was diluted in the BSA/Triton-X/Azide
solution with 1% NGS and incubated for 48 h at 4°C. For double-labeling
experiments, the following rabbit polyclonal antibodies were used: rabbit anti-
GABA (1:1000–1:4000; Sigma), rabbit anti-calretinin (1:1000; Chemicon),
rabbit anti-calbindin (1:1000; Chemicon), and rabbit anti-tyrosine hydroxylase
(1:500; Chemicon). Appropriate dilutions of these antibodies were added to the
mouse anti-BrdU for approximately 24 of the 48-h incubation, except for rabbit
anti-calbindin, which was incubated for the full 2 days. The sections were next
incubated for 2 h at RT with goat anti-mouse Alexa 488 (1:4000; Molecular
Probes) and goat anti-rabbit Alexa 546 (1:2000; Molecular Probes). Secondary
antibodies were diluted in BSA/Triton-X/Azide with 1% NGS. Sections were
counterstained with bis-benzamide, and cover-slipped with Mowiol + PPDA to
Quantification of immunocytochemically labeled OB interneurons
P21 brains, harvested from animals receiving BrdU at E14.5, were serially
sectioned into 4 separate horizontal series spanning the entire dorsal–ventral
axis of the OB. Three sets of four series (one series for each marker), collected
from three independent animals, were used. 5 evenly spaced sections from each
series were chosen for photo-documentation and counting. Montages were
constructed in Adobe Photoshop, and lines representing laminar divisions were
drawn on each montage. On each section, 6 non-overlapping radial probes
(ventricular to pial surface) were made along the anterior–posterior axis of the
OB (see Fig. 2A). In each probe, BrdU+cells in each laminar division were
counted. Double-labeled BrdU+cells were also counted in each layer. The total
numbers of BrdU+cells and BrdU-double-labeled cells, for each layer, were
summed across all three brains and percentages of BrdU+cells co-expressing
each marker calculated.
We adapted an approach described previously (Polleux and Ghosh, 2002),
to establish our E14.5 whole-telencephalon cultures. E14.5 EYFP+embryos
were sorted from wild-type littermates and transferred to ice-cold complete
Hank's balanced salt solution (complete HBSS) without sodium bicarbonate
but with 9 mM HEPES and 0.001% phenol red at pH = 7.4. Brains were
dissected in ice-cold complete HBSS. After removing the pia from wild-type
forebrains, cuts were made with a microscalpel blade to facilitate flattening of
each telencephalon. Cortices were cut into approximate thirds: one cut was
made above the OB, and two dorsal cuts were made at opposite poles of the
cortical hem (see Fig. 3A). Two cuts flanking the rostral and caudal aspects of
the septum were also made. These hemi-forebrains were transferred to laminin/
poly-lysine-coated 6-well inserts (Falcon) and oriented with their ventricular
surfaces facing upwards. LGE and MGE grafts, as well as grafts from the OB
and ventral spinal cord, were dissected from EYFP+brains and stored
separately in ice-cold complete HBSS. Fine forceps were used to make small
openings in either the LGE or MGE, and EYFP+LGE or MGE grafts were
transferred to a host telencephalon with a micropipette. After grafts were
positioned, the ventricular surface of cut forebrains was filled with complete
HBSS to unfold cortical and septal flaps and flatten them upon the filter surface.
These cultures were grown for 4 days at 37°C/95% O2/5% CO2in 1.8 ml of
Slice Culture Medium (as described previously) supplemented with 5% heat-
inactivated horse serum (Sigma) and 10 mM HEPES; media were exchanged
after 2 days.
388 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
Mesenchymal/Epithelial co-cultures were prepared as previously described
(LaMantia et al., 2000). E9.5 EYFP+ventrolateral forebrain was microdissected,
and frontonasal mesenchyme was removed by enzymatic digestion. This
epithelium was directly transplanted into E14.5 LGE, cultured alone, or with
wild-type frontonasal mesenchyme. Explants were grown floating on Millipore
filters in DMEM/10%FBS for 2 days at 37°C/95% O2/5% CO2. After 2 days in
vitro, isolated forebrain epithelium or forebrain epithelium co-cultured with
wild-type frontonasal mesenchyme was transplanted into the E14.5 LGE. The
M/E co-cultures were enzymatically digested, and the mesenchyme removed
prior to grafting.
Whole-telencephalon cultures were fixed at RT for 2 h or overnight at 4°C
with 4% paraformaldehyde in PBS. Tissue was rinsed in PBS and blocked in
permeability solution (Polleux and Ghosh, 2002) with 5% normal goat serum
(NGS) overnight at 4°C. Rabbit anti-GFP (1:2000; Molecular Probes) and
mouse primary antibodies were applied overnight at 4°C. After rinsing in PBS,
goat anti-rabbit Alexa 488 (1:4000; Molecular probes) and goat anti-mouse
Alexa 546 (1:2000; Molecular probes) were applied simultaneously overnight at
4°C. Tissue was rinsed in PBS, cleared in hypaque (Amersham) and mounted
onto slides. The following primary antibodies were used with the rabbit anti-
GFP antibody: mouse anti-Tuj1 (1:1600; Covance); mouse anti-Calretinin
(1:1000; Chemicon); mouse anti-GAD-6 (1:500; Developmental studies
hybridomabank); mouse anti-MAP-2(1:500; Sigma); mouse anti-neurofilament
165 (2H3) (1:5000; Developmental studies hybridoma bank); rabbit anti-GABA
(1:4000; Sigma) was used alone.
Quantification and statistical analysis of migration
For all experiments, seven independent whole-telencephalon preparations
wereevaluatedquantitativelyfor each transplantconfiguration. Forgraftsplaced
in the LGE, three non-overlapping fields in the OB and cortex, equidistant from
the graft site, were imaged with a confocal microscope. Similarly, three non-
overlapping fields in the OB and dorsal LGE (equidistant from the MGE graft
site) were imaged for grafts placed in the MGE. Each field represented a 20 μm
z-stack collected at an interval of 1 μm with a 40× objective. Confocal z-stacks
were maximally projected, and EYFP+cells were counted in each field. For each
experiment, the total numbers of EYFP+cells were recorded for each field and
analyzed by two-way ANOVA. The independent variables used in our two-way
ANOVA analyses were (1) the transplant configuration (homotopic versus
heterotopic) and (2) the target destination (OB versus Cortex; OB versus dorsal
LGE). All reported P values represent the statistical probabilities of an
interaction between these two independent variables.
Macroscopic images, bright field and fluorescent, of live or fixed
preparations were collected with a 4 Mega pixel Nikon digital camera mounted
on a Leica MZFLIII dissecting microscope. Fluorescently labeled cryosections
were imaged on a Leica DMR microscope at 20× or 40×. Epifluorescent images
were acquired with a cooled CCD camera (Hamamatsu ORCA) using OpenLab
Fig. 1. Cells labeled with BrdU on E14.5 migrate into the developing OB. (A) BrdU+cells are present in the OB rudiment (above dashed line) and along the anterior
horn of the lateral ventricle (arrowheads) after 6 h. (B) One day later, BrdU+cells accumulate in the OB and along the rostral extension of the lateral ventricle
(arrowheads). (C) After 3 days, BrdU+cells continue to fill the core of the OB as it enlarges. (D) By P0, BrdU+cells occupy the nascent glomerular (asterisks) and
granule cell (arrow) layers of the OB. (E) BrdU+cells are present in a continuous stream (arrowheads) extending from the anterior margin of the lateral ventricle to the
OB rudiment at E16.5. (F–G) Nucleic acid counterstain (F; bis-benzamide) reveals that BrdU+cells fill a cell-dense pathway (G) resembling the adult rostral migratory
stream. Scale bar in panels A and E = 500 μm, scale bar in panel A applies to panels B–D. OB = olfactory bulb; Ctx = cortex; RMS = rostral migratory stream.
389E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
(Improvision) image acquisition software. Fluorescently labeled whole-mount
preparations were imaged on a Zeiss LSM 510 upright confocal microscope
(UNC Neuroscience Center Confocal Microscopy Core) equipped with Argon
and Green HeNe lasers. Red and green fluorophores were simultaneously
collected on separate channels. For low-power montages, maximum projections
were made from 6-μm serial optical sections collected with a 10× Plan-Neofluar
objective. For high-power images, 0.5–1 μm serial optical sections were
collected with either a 40× Plan-Neofluar or a 63× Plan-Apochromat oil
immersion objective with up to 2× digital zoom. Images were adjusted for
brightness, contrast, and intensity in Adobe Photoshop.
Early genesis and migration of ventral forebrain cells to the
Birth dating studies suggest that most granule and periglo-
merular cells in the mouse OB are generated between E18 and
P5 (Hinds, 1968a). Nevertheless, there is significant evidence
that LGE precursors produce OB interneurons before E18
(Toresson and Campbell, 2001; Wichterle et al., 2001; Stenman
et al., 2003; Yun et al., 2003; Yoshihara et al., 2005). Thus, we
used BrdU pulse/chase labeling to determine whether a
significant population of cells is generated in the E14.5 ventral
forebrain and migrates to the OB between midgestation and
birth (Fig. 1). We counted the number of heavily labeled BrdU+
cells in non-overlapping radial probes of the OB in 3 separate
horizontal sections at each time point. 6 h after injection, BrdU+
cells are most likely generated within the OB rudiment (1314
cells/mm2; Fig. 1A). These cells may represent late-born mitral
or early-born tufted cells (Hinds, 1968a). At E15.5, the density
of BrdU+cells in the OB increases (1556 cells/mm2; Fig. 1B),
suggesting that a subset of labeled cells from distal locations
have migrated into the OB. The density of BrdU+cells increases
to a maximal level at E16.5 (2603 cells/mm2). This suggests
that the remaining E14.5-labeled BrdU+cells that will migrate
to the OB have arrived by this time (see also Fig. 3). As the
olfactory bulb continues to enlarge, the density of BrdU+cells
declines (E17.5: 1728 cells/mm2; Fig. 1C and P0: 1261 cells/
mm2; Fig. 1D). By P0, BrdU+cells are predominately located in
the nascent granule and glomerular layers of the OB (Fig. 1D).
To assess potential sources of this apparently migratory
population of BrdU+cells, we examined sagittal sections
through the developing forebrain. By E16.5, E14.5 BrdU+cells
are found in a continuous cell-dense subventricular stream
extending from the dorso-lateral LGE to the OB (Figs. 1E–G;
arrowheads, Figs. 1A–B). Apparently, a relatively large
population of cells, generated on E14.5, migrates in a distinct
pathway that may represent the presumptive rostral migratory
stream and accumulates in the nascent glomerular and granule
cell layers of the OB.
Identity and stability of early generated cells in the OB
Early generated cells in the OB may be transient, biased
toward a particular laminar fate, or contribute to molecularly
distinct subsets of interneurons. E14.5 BrdU+cells persist in the
granule and glomerular layers of P21 and P60 mice (Figs. 2A–
B), suggesting that at least some of these cells survive an early
postnatal wave of apoptosis in the OB (Fiske and Brunjes, 2001;
Saito et al., 2004). To better define the contribution of E14.5-
generated cells to distinct neuronal populations, we evaluated
the frequency and molecular identity of these cells in each layer
of the mature OB. E14.5 BrdU+cells occupy all laminae at P21
(Figs. 2A, D, G); the majority (80%), however, are located in
the glomerular and granule cell layers (Fig. 2C). Apparently, the
contribution of early generated cells to glomerular and granule
layer histogenesis exceeds previous estimates (Hinds, 1968a;
We next analyzed the expression of 4 molecular markers of
distinct interneuron classes: GABA, calretinin, calbindin, and
tyrosine hydroxylase (TH). 28% (856 double-labeled cells/3019
BrdU+cells) of E14.5 BrdU+cells in the glomerular layer are
double-labeled for GABA (Figs. 2D, E, R), 13% (251/1945) for
calretinin (Figs. 2G–I, R), 8% (264/3447) for calbindin (Figs.
2L–N, R), and 5% (196/4127) for TH (Figs. 2O–Q, R). 53%
(2387/4529) of BrdU+cells in the granule cell layer can be
labeled for GABA (Figs. 2D, F, R), while only 3% (128/3770)
express calretinin (Figs. 2G, J–K, R). BrdU-calretinin double-
labeled cells in the mitral cell layer (9%; 41/474) may represent
subsets of later-generated mitral cells (Qin et al., 2005) or
displaced granule or short axon cells—especially since over
half of the E14.5 BrdU+cells in the mitral layer co-label for
GABA (337/628; Fig. 2R). Thus, a subset of molecularly
diverse OB interneurons is generated on E14.5 and is retained at
least through the third week of postnatal development.
Specific migration of cells from the LGE to the OB
Genetic loss-of-function and in utero fate mapping studies
clearly implicate the LGE as the primary extrinsic source of OB
interneurons. Nevertheless, the initial migration of early
generated cells from the LGE to the rudimentary OB has not
been directly visualized or manipulated experimentally. Thus,
we developed an organotypic culture assay that preserves the
geometry of the developing telencephalon and facilitates
visualization of migratory cells as well as embryological
Fig. 2. E14.5 BrdU+cells are retained in the postnatal OB and co-label with OB interneuron markers. (A) BrdU+cells are located throughout the OB at P21 (blue = all
nuclei, green = BrdU). Box represents a single radial probe used for quantification (see Methods) and subsequent images. (B) BrdU+cells are retained in a similar
distribution in the P60 OB. (C) Distribution of BrdU+cells in the P21 OB. (D) GABA (red) and BrdU (green) labeling in the P21 OB. (E–F) BrdU-GABA double-
labeled cells (arrows) in the glomerular (E) and granule cell (F) layers. (G) Calretinin (CR; red) and BrdU (green) labeling in the P21 OB. (H–K) BrdU-CR double-
labeled cells (arrows) in the glomerular (H, I) and granule cell (J, K) layers. (L) Calbindin (CB; red) and BrdU (green) labeling in the glomerular layer of the P21 OB.
(M–N)BrdU-CB double-labeled cells(arrows). (O) Tyrosine hydroxylase (TH;red) and BrdU(green) labelingin the glomerular layer of the P21 OB. (P–Q) BrdU-TH
double-labeled cells (arrows). (R) Percentage of BrdU+cells that co-label for GABA, CR, CB, and TH, in each layer of the OB at P21. Scale bar in panel A = 500 μm;
scale bars in panels B, D, G, = 100 μm; scale bars in panels E, H = 25 μm. Scale bar in panel E applies to panel F; panel H applies to panels I–K, M–N, P–Q.
ONL = olfactory nerve layer; GL = glomerular layer; EPL = external plexiform layer; MCL = mitral cell layer; GCL = granule cell layer.
390 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
analyses of precursor potential and fate (Figs. 3A, B). After 4
days in vitro, homotopically transplanted LGE cells migrate
robustly from the rostral margin of the LGE in coherent streams
that converge at the base of the OB rudiment (Figs. 3C–E),
comparable to that seen for E14.5 BrdU+cells in vivo at E16.5
(see Fig. 1E). In the migratory path, these cells usually have
391 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
small, fusiform cell bodies with leading processes that end in
lamellate growth cones (Fig. 3F, arrowheads). The robust
migration of homotopically transplanted E14.5 LGE cells into
the OB is consistent with our BrdU data; nevertheless, our in
vitro assay spans several days, and it is possible that migratory
LGE cells are generated long after isolation and engraftment on
E14.5. To determine if substantial numbers of migratory LGE
cells destined for the OB are born on E14.5, we pre-labeled
Fig. 3. E14.5 LGE-derived cells robustly migrate to the OB. (A–B) Donor (EYFP) LGE tissue is isolated and grafted into the LGE of a host (WT) telencephalon. (C)
Fluorescence image of a living preparation grown 4 days in vitro (DIV); EYFP+cells migrate rostrally from the LGE graft site in a coherent stream (arrowheads). (D)
Confocal montage of a fixed whole-mount preparation, grown 4 DIV, and stained for EYFP (green) and Tuj1 (red). Large numbers of LGE-derived cells selectively
target the OB; migrating cells converge at the base of the OB rudiment. (E) Stream of LGE cells migrating rostrally (arrows) towards the OB. (F) LGE cells with
migratory morphologies found at a position similar to box in panel E. Arrowheads indicate lamellate growth cones on two of the migratory LGE cells. (G–I) E14.5
BrdU+LGE cells migrate into the OB rudiment. BrdU was administered in vivo at E14.5, and EYFP/BrdU-exposed LGE tissue was grafted homotopically. After 4
DIV, nearly half (301/625) of the EYFP+cells (green) found in the OB co-label for BrdU (red). Scale bars in panels D and G = 200 μm; scale bar in panel F = 25 μm;
scale bar in panel H = 50 μm. Asterisks = graft site; arrows in panel E indicate direction of migration; Wh-t = whole telencephalon.
392 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
Fig. 4. LGE-derived cells display cellular and molecular hallmarks of OB interneurons in the OB rudiment. (A–D) LGE cells display a range of morphologies in the
OB. (A–B) Some LGE cells have thin, unbranched apparent axons (arrowheads) and locally branched apparent dendrites. (C–D) Others have no morphologically
apparent axon-like processes, but have long, branched apparent primary dendrites. (E–P) Molecular identity of LGE-derived cells. EYFP+LGE cells (green) express
the early neuronal markers Tuj1 (E–G; red) and Map-2 (H–J; red), and the interneuron markers GABA (K–M; red) and calretinin (CR; N–P; red) in the OB rudiment.
Scale bar in panel D = 20 μm, applies to panels A–C; scale bar in panel E = 10 μm; scale bar in panel H = 25 μm, applies to panels K, N.
393 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
donor embryos with a 1-h BrdU pulse followed by a thymidine
chase, 2 h before harvesting and grafting. Approximately half
(48%; 301/625) of the LGE-derived EYFP+cells in the OB
were BrdU+(Figs. 3G–I). This reinforces our conclusion that
significant numbers of E14.5-generated LGE–derived cells
migrate specifically into the OB via a distinct pathway.
Differentiation of OB interneurons and projection neurons
A fundamental distinction among OB neurons is the
differentiation of an axon. Periglomerular cells have short
axons, granule cells lack axons entirely, and mitral cells develop
long axons that coalesce into the lateral olfactory tract (LOT).
Accordingly, we asked whether these cell biological distinctions
reflect intrinsic identities of LGE versus OB precursors. In the
OB rudiment in vitro, LGE-derived cells acquire a range of
morphologies (Figs. 4A–D), some of which closely resemble
mature OB interneurons. Some cells have multiple locally
branched dendrites with an apparent fine caliber short axon
(Figs. 4A, B, arrowheads) – similar to periglomerular cells –
while others have an apparent primary locally branched dendrite
with no recognizable axon (Figs. 4C, D)—cytological hall-
marks of granule cells. In the OB rudiment in vitro, most LGE
cells express Tuj1 (Figs. 4E–G) and Map-2 (Figs. 4H–J),
markers of immature neurons, as well as GABA (Figs. 4K–M),
the characteristic neurotransmitter of inhibitory interneurons. In
addition, a subpopulation of LGE cells expresses calretinin
(Figs. 4N–P), which labels both periglomerular and granule
cells in vivo. Therefore, cells generated from LGE precursors in
vitro at E14.5 acquire several diagnostic cytological and
molecular features of OB interneurons.
We next asked whether cells from the OB rudiment are
distinct from those in the LGE—particularly whether they,
unlike their LGE counterparts that migrate to the OB, generate
long axons. Cells in the OB rudiment of whole-telencephalon
cultures have axons that coalesce into a coherent bundle
resembling the LOTin vivo (Fig. 5A; Sato et al., 1998; Hirata et
al., 2001; Lopez-Mascaraque et al., 2005). To distinguish the
developmental potential of these cells versus those from the
LGE, we grafted EYFP+OB tissue into the OB or LGE. OB
cells do not appear to proliferate or migrate when grafted into
either the OB or LGE (Figs. 5B–F). These OB-derived cells
have irregularly shaped cell bodies with multiple neurites (Fig.
5E), even when grown in the LGE (Fig. 5D). Homotopically
Fig. 5. OB cells are morphologically and molecularly distinct from contemporary LGE cells. (A) A crystal of DiI (asterisk) was placed into the OB of a whole
telencephalon culture. Axons from labeled cells (inset) coalesce into a patent LOT. Coronal sections (1–3) of the DiI-labeled culture demonstrate the coherence of the
LOT (arrows) in the ventro-lateral forebrain, in vitro. (B) Homotopically transplanted OB graft cells partially fill the OB rudiment and extend long axons, after 4DIV.
(C) Cells (arrowheads) from OB graft do not migrate long distances when transplanted into the LGE. (D) High-magnification image of a differentiated OB-derived cell
in the LGE (box in panel C) with a single primary dendrite (arrow) and apparent axon (arrowhead). (E) OB cells (arrows), from homotopically placed OB grafts
(box in panel B), have irregularly shaped cell bodies and many neuritic processes. (F) High-magnification image of axon fascicles extending from OB graft (box
in panel B). (G–I) OB-derived graft cells (arrows) co-label for HuC/D, which labels mitral cells in the adult OB. Scale bars = 400 μm in panel B, 200 μm in
panel C, 50 μm in panels E, G. Width of panel D = 62 μm. Scale bar in panel E applies to panel F. Green = EYFP; red = DiI in panel A; Tuj1 in panel B;
neurofilament-165 in panel C; HuC/D in panels H and I. Asterisks = graft site; LOT = lateral olfactory tract.
394 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
transplanted OB cells generate axon fascicles that project away
from the OB rudiment (Figs. 5B, F). Many can be labeled for
HuC/D (Figs. 5G–I), a neuron-associated RNA-binding protein
seen selectively in mature mitral cells (Thompson Haskell et al.,
2002). Apparently, E14.5 OB cells are distinct from LGE-
derived cells. They express at least one mitral cell marker and
Fig.6. LGE andMGEcells havedistinctmigratoryspecificities. (A–B)Homotopicallytransplanted LGE cellsmigrate extensivelyinto the OB. (C–D) Heterotopically
transplanted MGE cells migrate dorsally from the LGE, avoiding the OB. (E) Quantification of migratory specificity. Top panel: EYFP+cells are counted in 3 non-
overlapping fieldsin the OB andCtx. Middlepanel:examplesof EYFP+LGE(left) andMGE (right)cells inthe OB and Ctx.Bottompanel: the numberof EYFP+cells
in the OB and Ctx is highly statistically different between LGE to LGE and MGE to LGE transplantation configurations (two-way ANOVA: P < 0.0001). (F)
Homotopically transplanted MGE cells migrate dorsally through the LGE and accumulate in the cortex. Inset = enlargement of box showing MGE cells in the cortex.
(G) LGE cells migrate short distances into the ventral forebrain when transplanted into the MGE and do not migrate in large numbers to either the cortex or OB. Inset:
enlargement of box showing LGE cells in the cortex. (H) Migration from the MGE was quantified as above. The number of EYFP+cells differs significantly between
MGE to MGE and LGE to MGE transplants (two-way ANOVA: P < 0.0001). Scale bars = 1 mm in panel A, 200 μm in panel B, 50 μm in panel E, and 400 μm in
panels F and G. Scale in panel A applies to panel C, panel B applies to panel D. Asterisks = graft site. Green = EYFP; red = GAD65in panels A and B, calretinin in
panels C and D, Tuj1 in panels F and G.
395 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
respond to local cues in the OB rudiment that promote axon
LGE and MGE cells have distinct migratory fates
Fate mapping studies indicate that migratory LGE and MGE
cells have distinct destinations (Anderson et al., 2001; Wichterle
et al., 2001). Local signals or cell autonomous factors may
determine the migratory capacity of LGE and MGE cells. To
assess these mechanisms, we evaluated the migratory capacity
and destinations of precursors grafted homologously or
heterologously into the LGE and MGE. Non-forebrain
precursors (e.g., E14.5 ventral spinal cord) incorporate in the
host LGE but do not migrate far beyond the margins of the graft
(data not shown). Apparently, the LGE environment does not
confer migratory properties on dramatically heterologous tissue.
In contrast, homotopically transplanted LGE cells migrate
extensively into the OB rudiment (Figs. 6A, B; see also Figs.
3C, D). We next asked if MGE precursors migrate to the OB
when placed in the LGE. MGE cells grafted into the LGE
undergo robust tangential migration into the cortex; however,
they almost completely avoid the OB (Figs. 6C, D). We
confirmed this quantitatively and observed approximately 16×
more LGE cells in the OB than the cortex in LGE to LGE
transplants; similarly, there were 18× more MGE cells in the
cortex than the OB for MGE to LGE transplants (Fig. 6E).
When we compared the numbers of cells in the OB and cortex in
each transplant configuration (two-way ANOVA: P < 0.0001),
we found a significant statistical difference in the migratory
destination of LGE versus MGE cells. Thus, migration of LGE
cells to the OB is robust, quantifiable, and statistically distinct
from that of MGE cells placed in the LGE environment.
We next asked whether LGE precursors behave similarly to
their MGE counterparts when placed in the MGE, or whether
they retain their migratory specificity for the OB. Homotopi-
cally transplanted MGE cells migrate to the cortex via routes
that approximate those described previously both in vivo and in
vitro (Fig. 6F; Anderson et al., 2001; Wichterle et al., 2001;
Polleux et al., 2002; Yozu et al., 2005). LGE cells, when grafted
ectopically in the MGE, primarily migrate dorsally into the
striatum; very few cells reach either the OB or the cortex (Fig.
6G, and inset). We confirmed this by comparing numbers of
cells in the dorsal LGE (dLGE) and OB in MGE to MGE and
LGE to MGE transplants. In both cases, we observed
significantly more cells in the dLGE than the OB (Fig. 6H;
17× difference for MGE to MGE, 8× difference for LGE to
MGE). The numbers of cells in the dLGE versus the OB in
MGE to MGE and LGE to MGE transplants were significantly
different (two-way ANOVA: P < 0.0001). Thus, LGE cells
placed in the MGE do not behave exactly like MGE cells, nor
do they access or fully execute their normal migratory program
to the OB.
It is possible that some of this apparent migratory specificity
reflects instructive or inhibitory signals from the OB, cortex, or
intervening migratory pathways. Direct apposition of LGE and
MGE tissue with their quantitatively dominant targets, OB and
cortex respectively, results in robust entry of migratory cells
(Fig. 7, upper left and lower right). Reversed pairing, however,
demonstrates a further distinction between LGE and MGE cells
(Fig. 7, lower left and upper right). LGE cells do not migrate in
substantial numbers into isolated cortical tissue, while MGE
cells readily migrate into isolated OB tissue. Thus, LGE cells
have a more limited capacity to enter potential forebrain targets
than MGE cells.
Developmental acquisition of migratory specificity
LGE versus MGE migratory specificity may follow the
morphogenetic and molecular differentiation of the ganglionic
eminences between E11 and E15 (Bulfone et al., 1993; Corbin
et al., 2000), or it may be established prior to the emergence of
distinct LGE and MGE compartments. Accordingly, we asked
whether ventral forebrain cells isolated from E9.5, 11.5, and
12.5 embryos – prior to or coincident with the emergence of the
LGE and MGE – migrate specifically to the OB when placed in
the E14.5 LGE (Fig. 8). E9.5 ventral forebrain epithelial (vFbE)
cells, presumably including LGE progenitors, do not appear to
migrate from the E14.5 LGE to any telencephalic location (data
not shown; see Fig. 8H; n = 10). In contrast, when explants of
E9.5-isolated vFbE are cultured intact for 2 days (Fig. 8A) and
then placed into the E14.5 LGE, migration is seen in
approximately 85% of our preparations (Figs. 8C, E, G;
n = 13); nevertheless, there is no apparent specificity of these
cells for the OB versus cortex. E9.5 vFbE cultured in the
presence of frontonasal mesenchyme (FnM; Fig. 8B), which is
Fig. 7. Target selectivity differs between LGE and MGE cells. Top row. when
directly apposed to their quantitatively dominant OB target (left), LGE cells
migrate from the graft and invade the adjacent tissue. When LGE cells are paired
with a cortical target (right), however, fewer LGE cells migrate from the graft
into the cortical target tissue. Bottom row. MGE cells, when paired with an OB
target (left), readily migrate into the inappropriate target tissue. When directly
apposed to a cortical target (right), they migrate extensively into their
appropriate target tissue. Scale bars = 400 μm. Scale bar in upper left applies
to lower left; scale in upper right applies to lower right. Asterisks = grafts.
396 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
normally apposed to the lateral ventral forebrain in vivo,
acquires an enhanced migratory capacity – without enhanced
migratory specificity – that approximates that seen from E11.5
LGE grafts (Figs. 8G, H). Thus, early ventral forebrain cells
autonomously acquire general migratory capacity between E9.5
and E11.5, prior to significant morphological differentiation of
the ganglionic eminences. Furthermore, extrinsic signals from
the frontonasal mesenchyme enhance this capacity.
Acquisition of LGE migratory specificity may proceed in
parallel with additional morphogenetic and molecular differen-
tiation of the LGE versus MGE. Morphogenesis of the MGE,
which begins around E11.5, precedes that of the LGE by
approximately 1 day. By E12.5, however, both structures are
easily discernable. E12.5 LGE cells migrate less robustly from
the E14.5 LGE environment than their E14.5 counterparts.
Nevertheless, they are more frequently seen in the OB than the
cortex (11× difference; Figs. 9A, C). In contrast, E12.5 MGE
cells migrate robustly into the cortex and few are seen in the OB
(8× difference; Figs. 9B, C). When the migratory behaviors of
homotopic E12.5 LGE and heterotopic E12.5 MGE cells are
compared (two-way ANOVA: P < 0.0001), they differ
significantly in their target preference, as is the case at E14.5.
Apparently, by E12.5, LGE cells have acquired their autono-
mous migratory preference for the OB and MGE cells have
adopted a cortical migration program.
The earliest population of OB interneurons is generated
primarily in the LGE, migrates into the rudimentary OB via a
distinct pathway, accumulates in the nascent glomerular and
granule cell layers, and differentiates into a cytologically and
molecularly diverse group of interneurons. A novel in vitro
assay allowed us to characterize the developmental specificity
of this pioneering population of OB interneurons for the first
time. The identity of their precursors reflects an apparent
balance of cell autonomous factors established or reinforced by
position in the LGE as early as E12.5, when the LGE becomes
morphologically and molecularly distinct. The specific migra-
tion of LGE cells to the OB depends upon their initial location
in the LGE. Cells from other locations can neither recognize nor
follow this migratory pathway. These positionally specified
LGE precursors and progeny likely constitute an antecedent of
the postnatal subventricular zone (SVZ) and rostral migratory
stream (RMS). Accordingly, cellular mechanisms that specify
the earliest OB interneurons may concurrently establish a
precursor niche that gives rise to migratory OB interneuron
neuroblasts throughout life.
Fig. 8. Ventral forebrain precursors autonomously acquire the ability to migrate
by E11.5. (A–B) EYFP+explants of E9.5 ventral forebrain epithelium (vFbE)
were cultured for 2 DIV, either alone, or with unlabeled frontonasal
mesenchyme (FnM:vFbE; arrowheads indicate mesenchymal tissue). (1) Bright
field, (2) fluorescent, and (3) merged images of explant cultures. Explants were
then grafted into the E14.5 LGE, grown for 4 DIV, and scored for migration
(0 = no migration; + = least migration, +++ = most migration). (C–F) vFbE cells
from vFbE (C, E) and FnM:vFbE explants (D, F) display a range of migration
when grafted into the E14.5 LGE. (G) Distribution of migration scores from
vFbE (2DIV) to LGE and FnM:vFbE (2DIV) to LGE transplants. (H)
Distribution of migration scores from similarly assessed E9.5 vFbE (uncultured)
to LGE and E11.5 LGE to LGE transplants. Scale bar in panel C = 200 μm,
applies to panels D–F. Green = EYFP; red = Tuj1. Asterisks = graft site.
397 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
Studying early OB interneurons in vivo and in vitro
Elegant in vivo lineage tracing experiments (Wichterle et al.,
2001) as well as more recent neurochemical and electrophys-
iological analyses (Xu et al., 2004; Butt et al., 2005) provide
insight into the derivation and differentiation of a broad range of
forebrain interneurons, including those in the OB. Our in vivo
BrdU labeling data extend and clarify many of these observa-
tions. Nevertheless, in vivo methods cannot easily generate the
quantity and uniformity of experimental data necessary to
evaluate precursor specificity, neuroblast migration, and inter-
mediate steps in neuronal differentiation. Our in vitro assay
allows rapid quantitative assessment of these mechanisms in the
midgestation telencephalon. We found that forebrain regional
architecture and development of long distance axon pathways is
preserved in these preparations (see also Seibt et al., 2003).
Using this assay, one can reproducibly and quantifiably vary the
position, stage, and derivation of precursors, and monitor their
subsequent migration and differentiation. Accordingly, we have
been able to evaluate directly the relationship between time,
position, and migratory specificity in the generation of OB
versus other forebrain interneurons.
Embryonic genesis of OB interneurons
The embryonic contingent of OB interneurons that we have
characterized has largely been ignored, perhaps because
previous studies indicate that most (>75%) are produced
postnatally (Hinds, 1968a; Altman, 1969; Bayer, 1983). Our
data likely underestimate the size of the earliest generated
population since we limit BrdU exposure to 1 h on E14.5.
Nevertheless, early generated cells are distributed widely in the
OB, present in all layers, and comprise a molecularly diverse
subset of GABA, calretinin, calbindin, and tyrosine hydroxy-
lase (TH)-expressing interneurons (Gall et al., 1987; Kosaka et
al., 1995; Kosaka et al., 1998) that are relatively insensitive to
an early wave of OB cell death (Fiske and Brunjes, 2001; Saito
et al., 2004). Accordingly, these cells may establish a stable
scaffold for prolonged postnatal addition of OB interneurons
(Altman, 1969; Bayer, 1983) and the corresponding construc-
tion of OB circuitry (Pomeroy et al., 1990). The prenatal
arrival of these cells may impact key aspects of early OB
development and subsequent function, including bulb growth,
which is compromised by mutations that reduce OB interneu-
ron numbers (Qiu et al., 1995; Anchan et al., 1997; Bulfone et
al., 1998; Toresson and Campbell, 2001; Long et al., 2003;
Yun et al., 2003), synaptogenesis, which begins at E15 (Hinds
and Hinds, 1976; Hwang and Cohen, 1985), and early
processing of odors, which is essential for initial feeding and
parental imprinting in rodents (reviewed by McLean and
The LGE is the primary source of early OB interneurons
We found that the LGE is the primary source of early
generated OB interneurons. It has been suggested that many
early OB cells – including interneurons – originate from
epithelial precursors in the rudimentary OB itself (Hinds,
1968b; Gong and Shipley, 1995). The persistence of some bulb
interneurons in Dlx1/2 mutants (Anderson et al., 1997), where
the primary genetic lesion presumably compromises migration
from the ganglionic eminences, seems consistent with this
conclusion. Nevertheless, our observations suggest that most
early generated OB interneurons are derived from the LGE,
while precursors from the E14.5 OB rudiment primarily display
cellular and molecular characteristics associated with mitral
cells. Accordingly, we suggest that OB morphogenesis relies on
Fig. 9. Migratory specificity is established by E12.5. (A) E12.5 LGE cells
transplanted into E14.5 LGE show limited migration; nevertheless, the cells
preferentially target the OB. (B) E12.5 MGE cells transplanted into E14.5 LGE
migrate dorsally into the cortex and avoid the OB, similar to E14.5 MGE cells.
(C) The average number of cells in the cortex and OB from E12.5 LGE to E14.5
LGE transplants is statistically different from E12.5 MGE to E14.5 LGE
transplants (two-way ANOVA: P < 0.0001). Scale bar in panel A = 500 μm,
applies to panel B. Green = EYFP; red = Tuj1 in panel A, calretinin in panel B.
Asterisks = graft site.
398 E.S. Tucker et al. / Developmental Biology 297 (2006) 387–401
the coordination of two sequential processes: one that first
specifies mitral cell precursors, presumably in the anterior
dorso-lateral telencephalon, at a fairly early stage (E9–10;
Jimenez et al., 2000; Nomura and Osumi, 2004), and one that
later regulates the production and migration of interneurons
from precursors in the LGE during midgestation. We found that
LGE cells acquire the general ability to migrate between E9.5
and E11.5 but do not migrate specifically to the OB until E12.5.
Therefore, this delay in OB migration may ensure the
appropriate temporal pairing of interneuron arrival with the
initial differentiation of postmitotic projection neurons in the
A unique migratory trajectory for OB interneurons
We provide direct evidence that a singular migratory
pathway is established between the LGE and rudimentary OB
during midgestation. LGE-generated neuroblasts selectively
recognize this pathway, while MGE-generated cells and other
heterologous cells do not. Previous observations indicate that an
organized migratory domain exists between the ganglionic
eminences and the rudimentary OB (Zerucha et al., 2000;
Pencea and Luskin, 2003). It was not clear, however, whether
this region constitutes a specific pathway for migratory cells
generated in the LGE. We found that most migratory LGE cells
do not stray far beyond the OB pathway, and most MGEcells do
not recognize this apparent antecedent of the RMS. Indeed,
LGE and MGE cells must travel in orthogonal trajectories
within this domain to reach their respective targets. LGE cells
seem to be more rigidly constrained in their migratory capacity
than MGE cells. MGE cells can enter both the OB and cortex
when placed directly next to either target, while LGE cells only
robustly enter the OB in the same configurations. Apparently, a
balance of attractive and inhibitory guidance cues, acting locally
within the ventral forebrain and in the appropriate target field,
establish distinct migratory trajectories for LGE and MGE cells.
Thus, LGE-generated neuroblasts may preferentially recognize
specific cues in the immature forebrain including Ig superfamily
molecules, integrins, and members of the neuregulin family,
whose expression remain enhanced in the adult RMS (Hu et al.,
1996; Chazal et al., 2000; Murase and Horwitz, 2002; Anton
et al., 2004).
Migratory specificity of LGE-generated neuroblasts
We have shown that LGE precursors are intrinsically and
autonomously programmed during a relatively brief period to
recognize a specific migratory pathway to the rudimentary OB.
Apparently, from this time onward, these cells retain this
specificity — indeed, E14.5 LGE cells placed in the adult SVZ,
migrate to the OB (Wichterle et al., 1999). The LGE
environment does not confer migratory specificity to other
cells during midgestation. LGE cells, however, must reside in
the LGE before migrating to the OB. Apparently, cell
autonomous identity reinforced by local signals is required for
LGE cells to migrate selectively to the OB. There is a critical
period between E11.5 and E12.5 when this selective migratory
capacity is established in the LGE. Before this time, general
migratory ability is acquired gradually, perhaps reflecting a
balance of intrinsic factors in the ventral forebrain epithelium
and extrinsic signals provided by adjacent frontonasal mesen-
chyme (LaMantia et al., 1993; Anchan et al., 1997; Xu et al.,
2005). Commitment to specific migratory fates appears to be
fairly rigid: once established, the migratory potential of LGE or
heterotopically transplanted MGE and CGE cells (Nery et al.,
2002; Yozu et al., 2005), as well as for LGE cells transplanted
into the MGE (Wichterle et al., 2001), and in each case, the
transplanted cells do not adopt migratory characteristics of their
host location. This suggests that precursors within the ventral
telencephalon acquire identities that not only define neuronal
subtypes (Fode et al., 2000; Parras et al., 2002; Schuurmans and
Guillemot, 2002) but also ensure migratory fidelity.
Initial and ongoing specification of OB interneurons
The genesis, migration, and differentiation of an initial
compliment of stable OB interneurons from positionally-
specified LGE precursors may represent the prenatal establish-
ment of the progenitor niche – the SVZ – and migratory
pathway – the RMS – for OB interneurons that persists through
adulthood (Altman, 1969; Luskin, 1993; Lois and Alvarez-
Buylla, 1994; Alvarez-Buylla and Garcia-Verdugo, 2002;
Haskell and LaMantia, 2005). During development, the
establishment of OB interneuron precursors in the LGE and
the patterning of a distinct migratory pathway to the OB may
represent two independent events: first, interneuron precursors
in the LGE autonomously acquire their specific identity, then,
LGE-derived neuroblasts uniquely recognize a migratory
pathway to the OB. Accordingly, a combination of position,
lineage, and selective migration during embryonic development
may define regions of the adult brain that mediate ongoing
replacement of OB interneurons. Our data show that these
events occur with great temporal and spatial specificity for LGE
precursors and their OB interneuron progeny. It remains to be
determined what aspects of this specificity are preserved
throughout life to facilitate the continued generation of OB
This work was supported by NIDCD postdoctoral fellowship
DC007047 to E.S.T, a NARSAD young investigator grant to
F.P., a NARSAD independent investigator grant to A.S.L.,
NINDS grant NS047701 to F.P., NICHD grant HD029178 to
A.S.L., and NIMH grant MH64065 to A.S.L. The UNC
Neuroscience Center Confocal Microscopy Core, which
provided a resource for confocal imaging, was supported by
NINDS grant NS031768. The authors thank Lance Brown,
Hunter Councill, and Josh Smith for their excellent technical
assistance, Clifford Heindel for mouse care and technical
support, and Amanda Peters for laboratory management.
Several antibodies used in this study were obtained from the
Developmental Studies Hybridoma Bank.
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