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The Journal of Neuroscience, January 15, 2003, 23(2):568-578
DLX5 Regulates Development of Peripheral and Central Components
of the Olfactory System
Jason E.
Long1, *,
Sonia
Garel1, *,
Michael J.
Depew1, *,
Stuart
Tobet2, and
John L. R.
Rubenstein1
1 Nina Ireland Laboratory of Developmental
Neurobiology, Department of Psychiatry, Programs in Biomedical
Sciences, Developmental Biology, Neuroscience and Oral Biology, Langley
Porter Psychiatric Institute, University of California, San Francisco,
San Francisco, California 94143-0984, and 2 The
Shriver Center, Department of Biomedical Sciences and Physiology,
University of Massachusetts Medical School, Waltham, Massachusetts
02452
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ABSTRACT |
Induction, neurogenesis, and synaptogenesis of the olfactory bulb
are thought to require interactions with the olfactory epithelium. The
Dlx family of homeobox genes is expressed in both the
olfactory bulb and olfactory epithelium. In particular,
Dlx5 is expressed in the olfactory placode,
olfactory epithelium, and local circuit neurons of the olfactory bulb.
Here we analyzed mice lacking DLX5 function. The
Dlx5
/ mutation reduces the size of
the olfactory epithelium. Although some olfactory neurons are formed,
they fail to generate olfactory axons that innervate the olfactory
bulb. Despite the lack of innervation, the olfactory bulb forms, and
neurogenesis of projection and local circuit neurons proceeds. However,
the mutation has a cell-autonomous effect on the ability of neural
progenitors to produce olfactory bulb local circuit neurons, with
granule cells more severely affected than periglomerular cells. In
addition, the mutation has a noncell-autonomous effect on the
morphogenesis of mitral cells.
Key words:
Dlx5; local circuit neuron; olfactory bulb; olfactory epithelium; tangential migration; GABA
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Introduction |
The olfactory bulb (OB) is an
evagination from the rostral telencephalon that receives primary
olfactory axonal innervation from neurons in the olfactory epithelium
(OE). The OB is a laminar structure (see Fig. 9A) (Shepherd,
1998
). Its outer nerve layer (ONL) consists primarily of olfactory
afferent axons and ensheathing glia. The axons from olfactory neurons
expressing a specific olfactory receptor converge, and synapse on, the
dendrites of glutamatergic projection neurons (mitral and tufted cells)
in structures called glomeruli. Interspersed between glomeruli are
local circuit neurons (periglomerular cells), which send their
dendrites into the glomeruli of the glomerular layer (PG). These
neurons are both GABAergic and dopaminergic [~85% of
tyrosine hydroxylase+ (TH) cells are also
GABA+ in the PG] (Gall et al., 1987;
Kosaka et al., 1995). Deep to the PG is the external plexiform layer
(EPL), which contains the horizontal processes of tufted and mitral
cells, the cell bodies of tufted cells, and the dendrites of a second
type of GABAergic local circuit neuron (granule cells). The next layer,
the mitral cell layer (MC), contains the cell bodies of the mitral
cells. These neurons grow their axons into the fibrous internal
plexiform layer (IPL). Below this fiber zone are the cell bodies of the granule cells, forming the granule cell layer (GC). Deep to the GC is
the subventricular zone (SVZ), a reservoir of progenitor cells that
produces new granule and periglomerular neurons in the adult brain
(Hinds, 1968a,b; Altman, 1969; Luskin, 1993; Lois and Alvarez-Buylla,
1994; Goldman and Luskin, 1998). At the center of the OB are ependymal
cells, the remnants of the neuroepithelial lining of the ventricle.
Development of the OB begins with its induction and evagination from
the rostral telencephalon. There is evidence that signaling from the
olfactory placode contributes to patterning the telencephalic anlage of
the OB (Graziadei and Monti-Graziadei, 1992; De Carlos et al., 1995;
LaMantia et al., 2000). Primary olfactory axons are also implicated in
regulating the early neurogenesis within the OB (De Carlos et al.,
1995; Gong and Shipley, 1995), although direct demonstration that
olfactory afferents are essential for OB neurogenesis is lacking. The
genesis and differentiation of OB projection and local circuit neurons
are under distinct genetic controls. The projection neurons (mitral and
tufted cells) have a pallial origin and are regulated by cortical
transcription factors such as Tbr1 (Bulfone et al., 1998),
whereas the local circuit neurons (periglomerular and granule cells)
have a subpallial origin and are regulated by transcription factors
such as Dlx1 and Dlx2 (Qiu et al., 1995; Bulfone
et al., 1998).
The Dlx genes are homeodomain transcription factors that
regulate development of multiple cell types derived from the
subcortical telencephalon (Qiu et al., 1995; Anderson et al., 1997;
Bulfone et al., 1998). Dlx1, Dlx2,
Dlx5, and Dlx6 are expressed in precursors of
telencephalic GABAergic and dopaminergic neurons. Their expression persists at lower levels in postmitotic neurons (Liu et al., 1997; Stuhmer et al., 2002a,b). In the telencephalon, expression of Dlx5 and Dlx6 generally occurs after
Dlx1 and Dlx2 (Eisenstat et al., 1999; Stuhmer et
al., 2002a,b).
Local circuit neuron development in the OB is sensitive to all
Dlx mutations studied to date. Both
Dlx1/ and
Dlx2/ mutants have a reduction in
TH+ neurons (Qiu et al., 1995) (J. E. Long and J. L. R. Rubenstein, unpublished
observations) and Dlx1&2/
mutants lack >95% of GABA+ and
TH+ neurons (Bulfone et al., 1998).
Dlx1&2/ mutants also lack
Dlx5 expression in most regions of the forebrain (Anderson
et al., 1997; Zerucha et al., 1997), and, therefore, it is possible
that the local circuit neuron defects in these mutants arise from DLX5
deficiency. To address this possibility, we investigated OB development
of Dlx5/ mutant animals.
Dlx5 is expressed in multiple components of the olfactory
system. It is expressed in the olfactory placode and in the OE; its
expression in the olfactory placode is required for morphogenesis of
the skeleton of the frontonasal prominence (Acampora et al., 1999;
Depew et al., 1999). Dlx5 is also expressed in the SVZ, GC,
and PG of the OB. Here we focus on the role of DLX5 regulating the
development of the OE and OB.
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Materials and Methods |
Animals and tissue preparation. A mouse mutant strain
with a null allele of Dlx5 was used in this study (Depew et
al., 1999). This mouse strain was maintained by backcrossing to
C57BL/6J mice for more than 10 generations. For staging of embryos,
midday of the vaginal plug was calculated as embryonic day 0.5 (E0.5).
Mouse colonies were maintained in accordance with the protocols
approved by the Committee on Animal Research at University of
California, San Francisco. Animals expected to contain
Dlx5/ mutant embryos were
killed by cesarean section. PCR was performed as described
previously (Bulfone et al., 1993; Depew et al., 1999) to genotype
offspring resulting from Dlx5 heterozygous matings. Heterozygous and wild-type embryos showed the same phenotype, so
both are used as controls throughout this paper. Embryos were anesthetized by cooling, dissected, and immersion fixed in 4% paraformaldehyde (PFA) in PBS for 4-12 hr. Samples were either cryoprotected in a gradient of sucrose to 30%, frozen in embedding medium (OCT; Tissue-Tek, Miles, Torrance, CA), and cut using a cryostat
or dehydrated in ethanol, embedded in paraffin, and cut using a microtome.
In situ hybridization. In situ hybridization
experiments were performed using 35S
riboprobes on 10 µm frozen sections as described previously (Bulfone
et al., 1993). We generously thank the following people for cDNAs: Drs.
Brian Condie (Medical College of Georgia, Augusta, GA)
(GAD67), Peter Gruss (Max Planck Institute of
Biophysical Chemistry, Gottingen, Germany) (Pax6),
Tom Curran (St. Jude Children's Hospital, Memphis, TN)
(Reelin), Dona Chikaraishi (University of Rochester,
Rochester, NY) (TH), and Francois Guillemot (Centre National de la Recherche Scientifique/Institut National de la Santé
et de la Recherche Médicale/Universite Louis Pasteur, Strasbourg, France) (Hes5). The Dlx1, Dlx2, and
Dlx5 plasmids were generated in the Rubenstein laboratory.
Histochemistry. Samples were sectioned at 10 µm and
mounted onto SuperFrost Plus slides (Fisher, Pittsburgh, PA). Sections were stained with either Gimori trichrome (E10.5) on paraffin-embedded sections or cresyl violet [E12.5 to postnatal day 0 (P0)] on
OCT-embedded sections and analyzed.
Bromodeoxyuridine labeling. Pregnant female mice were
injected intraperitoneally with 40 mg/kg bromodeoxyuridine (BrdU)
(Sigma, St. Louis, MO) and killed either 60 min later (see Fig. 7) or at E18.5 (see Fig. 8) as described previously (Anderson et al., 1997).
Immunohistochemistry. Immunohistochemistry was performed as
described previously (Marin et al., 2000). We used the following primary rabbit polyclonal antibodies: anti-NPY (diluted 1:3000; Incstar, Stillwater, MN); anti-GAD65 (diluted 1:1000; Chemicon, Temecula, CA); anti-GAD67 (diluted 1:2000; Chemicon); and anti-TBR1-c (diluted 1:100; kindly provided by Dr. M. Sheng, Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA). We used the following mouse monoclonal antibodies: anti-
-III tubulin (diluted 1:200; Promega, Madison, WI); anti-GAP43 (diluted 1:1000; Chemicon); and anti-REELIN (diluted 1:500; kindly provided by Dr. A. Goffinet, University of Louvain Medical School, Brussels, Belgium). We used the following rat monoclonal antibodies: anti-NCAM (diluted 1:100; Sigma); and anti-BrdU (diluted 1:10; Harlan,
Crawley Down, Sussex, UK). We used the following goat polyclonal
antibody: anti-OMP (olfactory marker protein) (diluted 1:2000; kindly
provided by Dr. F. Margolis, University of Maryland School of Medicine,
Baltimore, MD).
Cell counting. To estimate the total cell numbers in the
olfactory bulb, 10 µm coronal (transverse) cryostat sections were stained with 50 µg/ml Hoechst 33342 (Molecular Probes, Eugene, OR).
Wild-type olfactory bulbs had 82 ± 5 sections (n = 25 embryos), whereas the Dlx5/
mutant olfactory bulbs had only 48 ± 4 sections
(n = 25 embryos). Then, the number of cells within a
560 × 80 µm area of the wild-type and
Dlx5/ mutant olfactory bulbs was
counted. This area extended from the olfactory ventricle to the surface
of the olfactory bulb. The wild types (n = 4) had
593 ± 24 cells, whereas the
Dlx5/ mutants (n = 4)
had 606 ± 37 cells. Thus, the overall cell density within the
wild-type and Dlx5/ mutant olfactory
bulb was determined to be similar. However, because the length of the
Dlx5/ mutant olfactory bulb is ~60%
that of the wild type, we estimate that the total number of cells in
the Dlx5/ mutant olfactory bulb is
~60% that of the wild type.
To estimate the relative numbers of GAD67+
and Reelin+ cells in wild-type and
Dlx5/ mutant embryos, silver grains
were counted on sections from in situ hybridization using
GAD67 and Reelin probes. For
GAD67+ cells, silver grains were counted
in the granule cell and periglomerular layers. Generally, the amount of
GAD67 label in the Dlx5/
mutant olfactory bulb was asymmetric; lateral-superior parts had the
least labeling, and medial-inferior parts had the most. In the granule
cell layer, there were 1854 ± 62 grains in the wild-type sections
(n = 4) and a range of 621 ± 23 (lateral bulb) to
1228 ± 37 (medial bulb) grains in
Dlx5/ mutant embryos
(n = 4). Thus, there is a 33-67% reduction in the
number of silver grains in the granule cell layer of the
Dlx5/ mutant embryo. In the
periglomerular layer, the wild-type embryo had 1310 ± 23 grains
(n = 4), whereas the
Dlx5/ mutant had a range of 248 ± 41 (lateral bulb) to 1095 ± 11 (medial bulb) grains
(n = 4). Thus, there is a 16-82% reduction in the number of silver grains in the periglomerular layer of the
Dlx5/ mutant embryo. In the mitral
cell layer, there were 1265 ± 24 grains in the wild-type embryo
(n = 4) and 1642 ± 37 grains in the
Dlx5/ mutant embryo (n = 4). This is an ~30% increase in the number of silver grains in the
mitral cell layer of the Dlx5/ mutant
embryo. Because the olfactory bulb is smaller in the
Dlx5/ mutant, the increased
Reelin hybridization signal in a given coronal section may
reflect increased mitral cell packing density and not an increase in
total mitral cells.
To count the number of S-phase cells,
BrdU+ cells resulting from a 1 hr pulse of
BrdU at E18.5 were counted in the ventricular-subventricular zones of
the olfactory bulb. There were 599 ± 16 BrdU+ cells in the wild-type
(n = 3 sections) and 1101 ± 33 BrdU+ cells in the
Dlx5/ mutant (n = 3 sections). Thus, there is an ~46% increase in the number of
BrdU-positive cells in the ventricular and subventricular zones in the
Dlx5/ mutant.
DiI labeling. P0 Dlx5/
mutants and their wild-type littermates were immersion fixed with 4%
PFA, and their brains were then removed and kept in fixative. Crystals
of the axonal tracer DiI (Molecular Probes) were placed into the
lateral olfactory tract (LOT) to retrogradely label mitral cells of the
OB. Crystals of similar shape and size (100-200 µm in diameter) were
used in homozygous mutant and wild-type littermate embryos. Brains were
kept in 4% PFA at room temperature for 2-3 weeks to allow the DiI to
diffuse. Subsequently, brains were embedded in 5% low-melting-point
agarose (FMC Bioproducts, Rockland, MA), and 100 µm coronal
sections were cut with a vibrating microtome (VT1000S; Leica, Nussloch,
Germany). Sections were counterstained with 50 µg/ml Hoechst
33342 (Molecular Probes) and mounted using Aquamount (Polysciences,
Warrington, PA).
Slice culture. Embryos were removed by cesarean section from
timed pregnant Dlx5 heterozygous female mice mated with
Dlx5 heterozygous male mice. Brains were dissected in
ice-cold Krebs' buffer as described previously (Marin et al., 2000)
and embedded in 5% low-melting-point agarose (FMC Bioproducts), and
250 µm sagittal sections were cut with a vibrating microtome
(VT1000S; Leica). Slices were then placed onto transwell membranes (8 µm pore size, 24 mm diameter membrane; Costar, Acton, MA) that were coated previously with 1 mg/ml Vitrogen (Cohesion, Palo Alto, CA) for 1 hr. The transwell membranes containing the slices were placed into
Neurobasal media containing 2% B-27 supplement (Invitrogen, Gaithersburg, MD), 0.5% glucose, 2% glutamine, and 2%
penicillin-streptomycin. The slices were then injected with a
LacZ-encoding defective retroviral vector using a
micromanipulator and nitrogen injector (Marin et al., 2000). The
slices were placed in a 37°C incubator with 5% CO2 for 72 hr. The slices were then processed for
-galactosidase histochemistry as described previously (Marin et al.,
2000).
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Results |
Dlx5/ mutants have a
hypoplastic olfactory epithelium that fails to form normal axonal
connections with the olfactory bulb
Dlx5 is expressed throughout the olfactory placode and
later, in the olfactory pit, in addition to expression in other regions of the embryo (Fig.
1A). Its expression in
the olfactory placode is essential for development of the underlying
frontonasal prominence (Acampora et al., 1999; Depew et al., 1999).
Additional analysis of olfactory placode derivatives shows that the
Dlx5/ mutant olfactory pit is small
and lacks thickening of the medial epithelium at E10.5 (Fig.
1B,C). At later stages, the OE and
vomeronasal organ are greatly reduced in size (Figs.
1D-I,
2A-H,
3A-D; and data not
shown). The small neuroepithelium exhibits some normal molecular
characteristics of differentiation, including the presence of -III
tubulin and NCAM proteins at E12.5 (Fig. 2A-D),
expression of the mutant allele Dlx5m (a
transcript continues to be produced), Lhx2, Otx1,
Otx2, and Pax6 RNA at E13.5 (data not shown) and
expression of OMP at E18.5 (Fig. 2E-H).
Although some olfactory axons appear to grow from the neuroepithelium
and fasciculate (Fig.
2B,D,F,H,
arrowheads), these have not been detected contacting the OB
in the Dlx5/ mutant. This is further
demonstrated by the lack of GAP43 and OMP expression on the surface of
the main and accessory OBs at P0 (Fig. 2I-L),
analysis of Nissl-stained sections at E14.5 and E18.5 (Fig.
3A-D), and DiI axon tracing from the OE at P0 (data not
shown). The cribiform plate in the
Dlx5/ mutant has a paucity of foramina
for the passage of olfactory axons through the skull en route to the OB
(Fig. 3B,D; and data not shown),
further suggesting that few, if any, OE axons pass through the
cribiform plate or even contact the OB. These results indicate that the
Dlx5/ mutation results in a
hypoplastic OE that fails to produce axons that contact the OB.
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Figure 1.
Analysis of OE development in E10.5 Gimori-stained
(B, C) and E12.5 Nissl-stained
(D-I) control and
Dlx5/ mutant embryos.
A, Schematic of Dlx5 expression in an
E10.5 embryo (brain expression is not shown). B,
C, The olfactory pit is smaller in the mutant
(C). In addition, the size of the lateral
frontonasal process and medial frontonasal process are reduced. The
hole in the mutant section (C) is a sectioning
artifact. D-I, Series of horizontal sections from
rostral (D, E) to caudal (H,
I) illustrating the reduction of OE size in the
Dlx5/ mutant embryo (E, G,
I) compared with control (D, F,
H). Note the large distance between the mutant OE and
the telencephalon. AER, Apical ectodermal ridge;
BA2, BA3, BA4, branchial
archs 2, 3, 4; LFNP, lateral frontonasal process;
LV, lateral ventricle; mdBA1, mandibular
branchial arch 1; MFNP, medial frontonasal process;
OP, olfactory pit; OV, otic vesicle;
POB, primordial olfactory bulb; Se,
septum; VNO, vomeronasal organ. Scale bar (in
I): B-I, 400 µm.
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Figure 2.
Molecular analysis of the OE
(A-H) and olfactory nerve
(I-L) in control and
Dlx5/ mutant embryos. Antibody
staining of -III tubulin (A, B)
and NCAM (C, D) in E12.5 embryos
reveals that, despite its small size, the mutant (B,
D) OE expresses markers of differentiation. However,
development of the lateral OE is dramatically reduced
(B, D) compared with controls
(A, C). Arrowheads
show fasciculated axons leaving the OE. Antibody staining for mature
olfactory neuronal markers was performed using an antibody to OMP at
E18.5 (E-H). The mutant OE expresses OMP
(F, H) but in a much small region
of the OE compared with controls (E, G).
G and H are high-power magnifications of
E and F; arrows mark the
extent of OMP expression, and arrowheads mark
fasciculated axon bundles. H-J, Expression of GAP43
(J) and OMP (L) is absent
from the most superficial layer of the OB in the
Dlx5/ mutants, suggesting that the
olfactory nerve does not reach the OB. l, Lateral;
m, medial. Scale bar (in L):
A-D, G, H 175 µm;
E, F, I-L, 500 µm.
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Figure 3.
Nissl-stained sagittal sections of the
telencephalon and OE from E14.5 (A,
B) and E18.5 (C,
D) control and
Dlx5/ mutant embryos. At E14.5, it
is already apparent that the Dlx5/
mutant OB (B) is reduced in size when compared
with controls (A). In addition, there is also no
OE in this embryo, which occurs in 25% of the cases. At E18.5, the
reduction in OB size is more apparent in
Dlx5/ mutant embryos
(D). The mutant OE is greatly reduced in
size (D). Additionally, there are few, if
any, foramina in the cribiform plate of the mutant
(D). The asterisks in
B and D indicate the space between the
brain and cribiform plate that would normally be occupied by the
olfactory nerve. AOB, Accessory olfactory bulb;
CP, cribiform plate; Cx, cortex;
LV, lateral ventricle; NS, nasal sinus;
ON, olfactory nerve; Se, septum. Scale
bar (in D): A-D, 310 µm.
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Reduced GABAergic neuron production in the olfactory bulb of
Dlx5/ mutants
Dlx5 expression is prominent in the subcortical
telencephalon and its extension into the OB (Figs.
4C,
5A,C,E).
Although loss of DLX5 function does not appear to have a major effect
on development of the basal ganglia (Figs. 3A-D,
5A-J; and data not shown) (Acampora et al., 1999), the
growth and histology of the OB are disrupted by this mutation, seen
clearly at E14.5 and more dramatic at E18.5 (Figs. 3A-D,
4A-P, 5A-R). Nissl staining at E18.5
shows that Dlx5/ mutants lack the
distinct neuronal layers characteristic of the OB (Figs.
3C,D,
4I,J) (see Fig.
7E,F). Coronal sections also
suggest that the olfactory ventricle is smaller in the mutant (Figs. 2, 4, 6).
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Figure 4.
In situ RNA hybridization and
immunofluorescence analysis of the expression of several local circuit
neuron (A-H, K-N) and projection
neuron (O, P) markers in coronal
hemisections of E18.5 OBs. In control embryos, several layers are
apparent in the OB by Nissl stain, including the PG, MC, GC, SVZ, and
VZ (I). However, in the
Dlx5/ mutant, these layers are not
readily visible (J). In
situ analysis of Dlx1 and Dlx5 in
the wild-type embryo (A, C) shows
high expression in the SVZ, weaker expression in the VZ, GC, and PG,
and very low expression in MC. In the mutant embryo (B,
D), analysis of these markers
(Dlx1 and Dlx5m)
reveals laminar organization similar to control embryos but at slightly
reduced levels in the GC and PG. GAD67 expression in
control embryos clearly demarcates the GC and PG with strong expression
in these local circuit neurons (E). In the mutant
embryo (F), we see a reduction in the thickness
of GAD67+ cells in the GC in addition
to a reduction in levels of the GC and PG. GAD65 immunofluorescence
labels the PG and GC in controls (G). The
Dlx5/ mutant has a severe
reduction in granule cell somal and granule cell dendritic
expression and a moderate reduction in the PG
(H). TH expression shows a
dramatic reduction in the GC and PG in the mutant embryo
(L) when compared with controls
(K). Another marker expressed by PG neurons,
Pax6, shows a slight reduction in the mutant
(N) when compared with the control
(M). Pax6 expression is
also slightly reduced in the SVZ (M,
N). Reelin, a projection neuron
marker, demonstrates a thickening of the MC in
Dlx5/ mutant embryos
(P). Scale bar (in
P): A-F, I-P, 590 µm; G, H, 215 µm.
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Figure 5.
Expression of Dlx5,
GAD67, and Reelin at E14.5 and E18.5 in
sagittal sections through the forebrain. In A,
B, G, and H, expression in
the LGE and MGE is continuous into the OB. In the
Dlx5/ mutant, there is a slight
reduction in Dlx5m and
GAD67 expression in the OB (A,
B, G, H).
Dlx5 and GAD67 expression is also
continuous from the septum to the OB (C,
D, I, J).
Insets in I and J show a
higher magnification of the OB at E14.5 revealing the reduction of
GAD67 expression in the
Dlx5/ mutant embryo. Analysis of
Reelin, a marker of the projection neurons of the OB,
shows virtually normal expression, despite the mutant OBs small size
(M-P). At E18.5, the decreased expression of
Dlx5m and GAD67 further
demonstrate the reduction of GC and PG in the
Dlx5/ mutant (E,
F, K, L). However, at
E18.5, Reelin expression is more diffuse in the
Dlx5/ mutant
(R) than in control (Q).
Cx, Cortex; MZ, mantle zone;
Se, septum. Scale bar (in R):
A-R, 870 µm; insets in
I, J, 1000 µm.
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Figure 6.
Cell non-autonomous defect in mitral cells
revealed by DiI retrograde tracing and immunofluorescence. Hoechst
staining of P0 embryos reveals a defect in laminar organization in the
Dlx5/ mutants
(B). DiI placed into the LOT retrogradely
labels mitral cells of the OB. In control embryos (C,
E), radially oriented mitral cell bodies and their
radially extended dendritic processes are visible. In the
Dlx5/ mutants (D,
F), however, the orientation of the mitral cells
is disorganized and their dendritic processes are small and disoriented
(as shown by arrowheads). TBR1 (nuclear,
G) and REELIN (proximal dendrite,
I) immunofluorescence shows the MC in controls,
which is disorganized in the mutant (H,
J). Colabeling of TBR1
(red) and REELIN (green) in mitral
cells is shown in (K, L). Scale
bar (in L): A, B, 775 µm; C-J, 320 µm; K,
L, 155 µm.
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OB laminar organization was further studied at E18.5 using in
situ hybridization on cross sections with RNA probes that define cell types in distinct layers (Fig. 4) (see Fig. 9A). For
the progenitor layers, we examined the expression patterns of the Dlx gene family members. Dlx1, Dlx2,
Dlx5, and Dlx6 are differentially expressed in
the progenitor and neuronal cell layers (Fig. 4A-D; and data not shown) (Dolle et al., 1992; Bulfone et al., 1998; Stuhmer
et al., 2002a). They are weakly expressed in the ventricular zone (VZ)
and strongly expressed in the SVZ. The expression patterns of
Dlx1, Dlx2, Dlx5m,
and Dlx6 in the Dlx5/
mutant suggest that the size of the SVZ, relative to the rest of the
OB, is increased.
To assess the development of OB local circuit neurons, we analyzed RNA
expression of the Dlx genes GAD67, GABA
transporter, TH, and Pax6 and protein
expression of GAD65 and TH (Fig. 4; and data not shown).
Dlx1, Dlx2, Dlx5, and Dlx6
are expressed in the GABAergic and dopaminergic neurons of the GC and
PG. The expression patterns of Dlx1, Dlx2,
Dlx5m, and Dlx6 in the
Dlx5/ mutant show that the GC and PG
are reduced (Fig. 4A-D; and data not shown) (see
Fig. 9). Expression of GAD67, one of the enzymes that makes
GABA, weakly marks the SVZ and strongly labels the GC and PG in the
normal OB (Fig. 4E) (Behar et al., 1994). In the
mutant, there is a prominent decrease in GAD67 expression, specifically, a decrease in the size and intensity of labeling in the
GC and a slight reduction in the PG (Fig.
4E,F) (see Fig. 9).
Expression of GAD65 protein is readily detectable in the GC and PG
(Fig. 4G) (Feldblum et al., 1993; Esclapez et al., 1994). In
the Dlx5/ mutant, there is a severe
reduction of GAD65 granule cell expression, including their projections
through the MC and EPL. In addition, the
Dlx5/ mutant shows reduced GAD65
expression in the PG (Fig. 4G,H) (see Fig.
9). Expression of TH marks a subset of granule and periglomerular dopaminergic cells (Gall et al., 1987; Kosaka et al., 1995; Toida et
al., 2000; Baker et al., 2001). In the
Dlx5/ mutant, expression of
TH is almost eliminated from the GC and is reduced in the PG
(Fig. 4K,L). Pax6 is
coexpressed with TH in periglomerular cells, is moderately
expressed in the SVZ, and is strongly expressed in the VZ (Fig.
4M,N) (Gall et al., 1987; Stoykova and Gruss, 1994; Kosaka et al., 1995; Dellovade et al., 1998;
Toida et al., 2000). Its expression in periglomerular cells is reduced
similar to the reduction of TH (Fig.
4M,N) (see Fig. 9),
consistent with their coexpression.
Estimates of olfactory bulb cell numbers were made on the basis of the
~40% reduction of olfactory bulb length in the
Dlx5/ mutant and the numbers of
Hoechst 33342-stained cells in wild-type and
Dlx5/ mutant olfactory bulb sections
(data not shown; see Materials and Methods). The cell counts revealed
an ~40% reduction in the total number of OB cells. The change in
cell number reflects the net result of decreases in granule and
periglomerular cells and increases in VZ-SVZ cells (for counting data,
see Materials and Methods). These results provide evidence that the
Dlx5/ mutation decreases the
differentiation of progenitor cells, which leads to decreased numbers
of GAD67+ and
TH+ OB local circuit neurons.
Analysis of the projection neurons of the OB was performed using
Reelin and Id2 expression. At E18.5,
Reelin expression is primarily restricted to mitral
cells (Bulfone et al., 1998). Although the MC lacks its prominence in
Nissl- and Hoechst-stained sections of the
Dlx5/ mutants (Figs.
3C,D, 4I,J,
6A,B,
7E,F),
Reelin labeling of these cells is robust, and the pattern
suggests that this layer is thicker than normal (Fig.
4O,P) (see Fig. 9). A similar result was observed with Id2 expression (data not shown) (Neuman et al., 1993).
The thickened mitral cell layer in the
Dlx5/ mutant may reflect increased
cell density, which could be caused by packing a normal number of
mitral cells into a smaller olfactory bulb.
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Figure 7.
Proliferation assays reveal a loss of ONL cells. A
1 hr pulse of BrdU given at E14.5 and E16.5
(A-D) labels mitotically active cells in the VZ,
SVZ, ONL, and mesenchyme (Mz) in and surrounding the OB.
The density of BrdU-labeled cells appeared normal in the VZ, SVZ, and
mesenchyme, whereas by E16.5, a decrease is apparent in the
Dlx5/ mutants most superficial
layer of the OB (arrowhead). A 1 hr pulse of BrdU at
E18.5 (I, J) shows a large
reduction in the number of BrdU-positive cells in the
Dlx5/ mutants most superficial
layer of the OB. Hes5 expression, which marks many types
of dividing cells, is also reduced in the
Dlx5/ mutants most superficial
layer of the OB (G, H). NPY
expression in olfactory nerve ensheathing glia is lost in the
Dlx5/ mutant (K,
L). Scale bar (in L): A,
B, 220 µm; C-F, I-L,
500 µm; G, H, 614 µm.
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Subcortical migrations of local circuit neurons are not appreciably
affected in the Dlx5/ mutants
To evaluate potential mechanisms underlying the
Dlx5/ mutant OB phenotype, we studied
the production of local circuit and projection neurons during
development using in situ hybridization on parasagittal sections (Fig. 5). For local circuit neurons, we used Dlx5
expression to assess progenitors and GAD67 expression to
assess postmitotic GABAergic cells. For projection neurons, we studied
the expression of Reelin.
At E14.5, there appears to be at least three subcortical SVZ zones that
are continuous with the SVZ of the OB; they are the SVZ of the septum,
lateral ganglionic eminence (LGE), and medial ganglionic eminence
(MGE). These zones are revealed by the expression of Dlx5
and GAD67 (Fig.
5A,C,G,I).
The lateral side of the OB is continuous with
Dlx5+ and
GAD67+ cells in the LGE and the MGE
(Fig. 5A,G). The medial side of the
OB is continuous with Dlx5+ and
GAD67+ cells in the septum (Fig.
5C,I). Dlx5- and
GAD67-expressing cells enter the ventral side of the OB.
These findings suggest that OB local circuit neurons are derived from
several subcortical sources, which may generate cells that tangentially
migrate into the OB via a ventral route.
The Dlx5/ mutation did not grossly
affect SVZ or mantle zone (laminas containing postmitotic
neurons and their processes) expression of
Dlx5m or GAD67 in the LGE, MGE,
or septum (Fig.
5B,D,H,J).
In addition, cortical GAD67+
neurons, which are also primarily derived from
Dlx-expressing subcortical sources (Marin and Rubenstein,
2001), were not appreciably reduced (Fig. 5G-J). On
the other hand, GAD67 expression in the mantle zone of the
OB was reduced (Fig. 5H,J).
This suggests that the early production of OB local circuit neurons is
defective. Although early production of OB local circuit neurons is
reduced, the generation of Reelin+
projection neurons appears normal at E14.5 (Fig. 5M-P).
In situ hybridization analysis at E18.5 also showed that
Dlx5m and GAD67 expression in
the OB SVZ appeared normal in Dlx5/
mutants, whereas the number of Dlx5+
and GAD67+ OB neurons was
reduced in the GC and PG (Fig.
5E,F,K,L).
Reelin expression in the MC remained robust, but its tight
laminar organization was disrupted (Fig. 5Q,R).
Thus, the Dlx5/ mutation causes the
decreased production of GAD67+ local
circuit neurons of the OB from early ages and, although projection neuron production appears normal, the lamination of the MC
is disrupted.
Non-autonomous defect in mitral cell morphogenesis in the
Dlx5/ mutants
Although Dlx5 is not expressed in mitral cells, and is
probably not expressed in mitral cell precursors, we observed a
noncell-autonomous effect on the MC in the
Dlx5/ mutant. As shown by Nissl and
Hoechst staining (Figs. 4I,J,
6A,B, 7E,F) and by
Reelin, Id2, and Tbr1 expression
(Figs. 4O,P, 5Q,R, 6G-L; and data not shown),
Dlx5/ mutants have a thickened MC
whose contour is irregular. To further evaluate this phenotype, we
retrogradely labeled the mitral cells and their dendrites with DiI. DiI
labeling in the LOT labeled the GC and IPL (consisting of mitral cell
axons), mitral cells bodies in the MC, and their processes in the EPL
and PG. In controls, mitral cells were regularly spaced and had
radially oriented dendrites, whereas in the
Dlx5/ mutants, the orientation of the
mitral cell dendrites was variable (Fig. 6C-F). This
result was confirmed by immunofluorescent double labeling of mitral
cells for TBR1 (Fig.
6G,H,K,L,
nuclear labeling) and REELIN (Fig. 6I-L, proximal
dendrite labeling). This analysis provides additional evidence that the
orientation of proximal dendrites was rotated, that the dendritic trees
were smaller, and that the mitral cells and their axons were in the
same layer (Fig. 6G-L). These results further support the
idea that the Dlx5/ mutation alters
the morphology of the mitral cells. The potential mechanisms underlying
this phenotype are considered in Discussion.
Analysis of proliferation and cell death in the olfactory bulbs of
Dlx5/ mutants
The in situ hybridization analyses suggest that
Dlx5/ mutants have a deficit in the
production of local circuit neurons that begins as early as E14.5. To
test whether this is attributable to a defect in the
proliferation of OB progenitors, we used a BrdU incorporation assay at
developmental stages when local circuit neurons constitute the majority
of neurons being generated (Hinds, 1968a,b, 1972a,b). One hour after a
pulse of BrdU at E14.5, E16.5, or E18.5, we identified the location of
cells in S-phase of the cell cycle in coronal sections of
Dlx5/ mutants and control littermates
(Fig. 7A-D,I,J).
At each of these ages, the BrdU pulse labeled three sets of cells: (1)
OB progenitors in the VZ and SVZ; (2) cells in the ONL; and (3)
mesenchymal cells surrounding the brain (i.e., meninges). In the
Dlx5/ mutants, the size of the
progenitor zone, and the density of S-phase cells within it, appeared
approximately normal, although we cannot rule out a subtle change in
the rate of proliferation in this layer.
In contrast, there was a reduction in the number of proliferating
cells in the most superficial layer of the OB in the
Dlx5/ mutant at E16.5 and E18.5 (Fig.
7C,D,I,J).
To confirm this finding, we used in situ hybridization with
Hes5, a basic helix-loop-helix gene that is expressed
in proliferating neural progenitors (Akazawa et al., 1992) and in cells
of the ONL (Fig. 7G). In controls, Hes5
expression was high in the VZ, moderate in the SVZ, and high in the
ONL, whereas in the Dlx5/ mutant,
Hes5 expression appeared normal in the OB progenitor zones
but was very low in the most superficial area of the OB (Fig.
7G,H). The proliferating cells in the ONL
are known to differentiate into specialized glia, a subset of which
express NPY (Ubink et al., 1994; Ubink and Hokfelt, 2000). These cells
are derived from the olfactory placode-epithelium (Doucette, 1993;
Mallamaci et al., 1996). Indeed, NPY expression in the ONL is greatly
reduced at E18.5 in the Dlx5/ mutant
(Fig. 7K,L). The residual
BrdU-labeled cells surrounding the mutant OB may correspond to
mesenchymal cells, such as the meninges (Fig.
7I,J). Thus, the
Dlx5/ mutation reduces the production
of olfactory nerve glia, probably attributable to its function in the
development of the OE.
To assess whether the decreased numbers of local circuit neurons could
be attributable to increased levels of cell death, in either the
progenitor or postmitotic cell populations, we used the terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling (TUNEL) apoptosis assay. We found similar levels of TUNEL+ cells in
Dlx5/ mutants and control littermates
at E14 and E18 (data not shown). These results demonstrate that the
Dlx5/ mutation does not have a
noticeable effect on cell death or proliferation using the methods that
we used. However, there is a dramatic reduction in the number of
BrdU-labeled cells in the most superficial layer of the
Dlx5/ mutant OB, which likely
correspond to olfactory nerve glia.
Analysis of neuronal migration in the olfactory bulb and rostral
migratory stream of Dlx5/
mutants
To assess whether the reduction in granule and periglomerular
cells is attributable to defects in migration, we used BrdU birth-dating and slice culture migration assays. BrdU pulses were performed on E14.5 and E16.5 embryos to label cells going through S-phase at that time. The BrdU-treated embryos were harvested on E18.5,
and the positions of BrdU-labeled cells were analyzed in coronal
sections of the OB. The majority of mitral cells are born before E14.5
(Hinds, 1968a,b, 1972a,b); therefore, labeled cells outside the
progenitor zones in this experiment will tend to correspond to local
circuit neurons, tufted cells, and glia (Hinds, 1968a,b, 1972a,b). BrdU
exposure at E14.5 and E16.5 in control embryos led to BrdU
incorporation into some cells that remained in the progenitor zones and
some that were located superficially in the OB (Fig.
8A-D). In the
Dlx5/ mutants, fewer BrdU-labeled
cells were present outside of the progenitor zone. This is particularly
clear for the embryos labeled at E16.5 (Fig.
8C,D). Furthermore, labeling at E14.5 resulted in
an accumulation of BrdU+ cells in the SVZ
(Fig. 8A,B, note the increased
density of labeled cells in the SVZ and the very small olfactory
ventricle), suggesting that the Dlx5/
mutation reduces the rate at which SVZ progenitors mature into neurons.
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Figure 8.
Migration assays in the
Dlx5/ mutant. A pulse of BrdU
given at E14.5 with survival until E18.5 shows a reduction in the
number of BrdU+ cells at the periphery of the OB in
the Dlx5/ mutant and an increase
in the density of BrdU+ cells within the SVZ
(A, B). A BrdU pulse at E16.5 with
survival until E18.5 shows a clear reduction in the number of
BrdU+ cells in the mantle zone of the OB
(C, D). A LacZ-encoding
replication incompetent retrovirus was injected into parasagittal
slices from an E18.5 telencephalon into multiple positions along
proximal positions of the RMS and then stained for
-galactosidase activity to identify the positions of
LacZ-expressing migratory cells (E,
F; shown by arrowheads).
Cx, Cortex. Scale bar (in F):
A-D, 445 µm; E, F, 1150 µm.
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The BrdU results suggest that radial migration from the progenitor zone
to the local circuit neuron layers is reduced in the Dlx5/ mutants, which could result from
either reduced neuronal production or a defect in migration. To assess
whether tangential migration along the rostral migratory stream (RMS)
is affected by the Dlx5/ mutation, we
used a slice culture cell migration assay (Tobet et al., 1996) (Fig.
8E,F). Parasagittal
vibrating microtome slices from E18.5 control and
Dlx5/ mutant embryos were generated.
We labeled the RMS with injections in five to six different locations
using a LacZ-encoding replication incompetent retroviral
vector. The slices were grown for 72 hr, and the locations of
LacZ-expressing cells were assessed by -galactosidase activity. The experiment showed that
Dlx5/ mutants have tangential
migration in their RMS (Fig.
8E,F) (n = 5 controls; n = 5 Dlx5/
mutants). Similar results were obtained using DiI labeling of migrating
cells (data not shown). These results provide evidence that tangential
migration still occurs in the Dlx5/
mutants. Additional studies are needed to determine whether the efficiency of migration is reduced.
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Discussion |
Herein we show that DLX5 has a central role in the development of
the primary structures involved in olfaction. Murine
Dlx5/ mutants have a hypoplastic OE
that fails to produce axons which innervate the OB. Despite the lack of
innervation, the OB still forms, although with a reduced size and
altered lamination. The size and lamination defects (Fig.
9) are contributed to by cell-autonomous defects in local circuit neuron production and by a noncell-autonomous effect on the orientation and dendritic branching of the mitral cells.
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Figure 9.
Diagrams showing OB laminar organization and
molecular expression in E18.5 wild-type (A) and
Dlx5/ mutants
(B). The top half of each OB shows
the laminar organization (each color representing a
different layer) and principal cell types. The bottom
half of each OB shows the expression of various genes, in which
increasing color density represents increasing levels of
expression. The top half of the mutant diagram
illustrates that Dlx5/ mutants
lack an ONL (blue), have a thicker MC (light
green), and appear to have a defect in the migration of SVZ
cells born at E14.5 (darker orange; see Fig.
8A,B). In addition, the
mutation disrupts the radial orientation of the mitral cells
(triangles) and their processes, reduces the numbers and
alters the processes of periglomerular and granule cells
(red and green circles), and virtually
eliminates glia in the ONL (yellow spindles). The
bottom half of the
Dlx5/ mutant diagram illustrates
the lack of an ONL, emphasized by the loss of OMP, GAP43, NPY, and
Hes5 expression, shows the reduced expression of
Dlx1, Dlx2,
Dlx5m, Dlx6,
TH, GAD65, and GAD67 in the GC and PG,
and the reduced expression of Pax6 in the PG. See figure
for color and shape definitions. V, Olfactory
ventricle.
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Loss of normal olfactory input to the telencephalon is not
essential for olfactory bulb neurogenesis
Previous studies have suggested that the olfactory placode and/or
epithelium may have a key role in the initiation of OB development and
neurogenesis (Graziadei and Monti-Graziadei, 1992; LaMantia et al.,
1993, 2000; Gong and Shipley, 1995). Although Dlx5 is expressed in the olfactory placode,
Dlx5/ mutants are able to produce an
olfactory pit and differentiate a small amount of neuroepithelium.
Thus, DLX5 is not essential for specification of the entire olfactory
placode. Perhaps, other Dlx family members (e.g.,
Dlx6) compensate for early DLX5 function in this
tissue; this possibility can be studied in
Dlx5&6/ mutants that have been
generated recently (Robledo et al., 2002).
The OE is severely hypoplastic in the
Dlx5/ mutants (Figs. 1-3). As early
as E10.5, Dlx5/ mutants show reduced
olfactory neuroepithelial structures (Fig. 1C). At this
point, it is uncertain whether the mutation causes a general hypoplasia
or whether specific subdivisions of the OE are preferentially affected.
Loss of Dlx5 also affects the differentiation of the
olfactory neurons. Although they can express NCAM, -III tubulin and OMP (Fig.
2B,D,F,H),
their axons fail to grow to the olfactory bulb. Although we cannot
completely rule out the possibility that a few OE axons do contact the
OB, our analyses of Nissl-stained, Hoechst-stained, OMP, GAP43, S100,
and calretinin immunohistochemistry, and DiI labeling of OE axons all
support the observation that no axons contact the OB. The mechanism(s)
underlying this defect could lie in either the olfactory neurons or the
environment through which they navigate, because DLX5 function is
required in the morphogenesis of frontonasal mesenchyme (Acampora et
al., 1999; Depew et al., 1999) and in the differentiation of the OB
(Figs. 3-9).
Associated with the OE and axonal abnormalities are the deficits in
olfactory ensheathing glia in the OB nerve layer and
gonadotrophin-releasing hormone (luteinizing hormone-releasing
hormone) neurons in the forebrain of the
Dlx5/ mutants (Fig.
7L,M; and data not shown). These
cell types are derived from the olfactory placode and migrate along the
olfactory nerve (Doucette, 1993; Tobet et al., 1993). These
deficiencies could be attributable to a role of DLX5 in the
differentiation of these cell types and/or attributable to a defect in
their migration that is secondary to the failure of olfactory nerve growth.
There is evidence that early olfactory axons may regulate cell cycle
kinetics in the telencephalic anlage of the OB (Gong and Shipley,
1995). Although we have not directly ruled out this possibility, the
fact that, in the absence of most olfactory axons, the OB is induced
and its early neurogenesis (mitral cells) appears normal suggests that
olfactory axons are not necessary for these processes. A similar result
is observed in Mash1/ mutants, which
fail to produce olfactory neurons (Guillemot et al., 1993; Casarosa et
al., 1999). However, Pax6sey/sey mutants
also lack olfactory axons but have molecular features of a
nonevaginated OB-like structure (Lopez-Mascaraque et al., 1998; Jimenez
et al., 2000). The mechanism underlying the failure of OB evagination
in Pax6sey/sey mutants may be secondary to
PAX6 function in the olfactory epithelium and/or the telencephalon
(Anchan et al., 1997).
Although olfactory axons are not essential for the induction and growth
of the OB, we provide evidence that they are required for its laminar
properties. Nissl and Hoechst staining of
Dlx5/ mutant OBs fail to reveal normal
laminar properties, despite the laminar expression of several GC, MC,
and PG markers (Figs. 4, 6, 7, 9). The lack of histological lamination
is not entirely attributable to the deficiencies in local circuit
neurons, because Dlx1&2/ mutants,
which lack local circuit neurons, exhibit clear OB lamination (Bulfone
et al., 1998). DiI labeling of mitral cells in the
Dlx5/ mutants shows that these cells
lack a radial orientation and have hypoplastic dendritic trees.
Reelin labeling shows that the mitral cell layer is thicker
(Figs. 4O,P, 9), perhaps attributable to
haphazard packing of mitral cell bodies (Fig. 6C-L). These non-autonomous effects, which may result from the lack of OE input, contribute to the defect in OB lamination in the
Dlx5/ mutants. Thus, olfactory axons
probably have a central role in organizing cellular morphogenesis of OB neurons.
Dlx genes regulate the generation of olfactory bulb
local circuit neurons
Dlx1, Dlx2, Dlx5, and
Dlx6 are expressed in the progenitors of OB local circuit
neurons (Figs. 4, 5, 9) (Liu et al., 1997; Stuhmer et al., 2002a).
Their expression is maintained in postmitotic granule and
periglomerular neurons, albeit at a lower level (Fig. 4, 9) (Stuhmer et
al., 2002a,b). Dlx2/ mutants have
greatly reduced numbers of TH+
periglomerular cells (~80%) (Qiu et al., 1995),
Dlx1/ mutants less so (~15%
reduction) (Long and Rubenstein, unpublished observations), whereas
Dlx1&2/ mutants lack most
GABA+ and TH+
neurons (Bulfone et al., 1998) (Long and Rubenstein, unpublished observations). Dlx1&2/ mutants fail to
express Dlx5 in the SVZ of most of the telencephalon; however, residual expression is found in part of the septum (Anderson et al., 1997; Zerucha et al., 1997). Through the analysis of the Dlx5/ mutants, we demonstrated that
the severe reduction of OB local circuit neurons in
Dlx1&2/ mutants is not attributable
only to the loss of Dlx5. In addition, we demonstrated that,
like Dlx2/ mutants,
Dlx5/ mutant mice have a hypomorphic
OB local circuit neuron phenotype (Fig. 9). The
Dlx5/ mutation appears to
preferentially affect granule cells as evidenced by the particularly
severe reduction of GAD67, GAD65, and
TH expression in this layer (Fig.
4E-H,K,L). The
reduction in TH expression could be caused by the lack of
olfactory axons (Baker et al., 1983; Baker, 1990; Baker and Farbman,
1993). However, we suggest that a cell-autonomous mechanism contributes
to this phenotype, because both Dlx2/
and Dlx1&2/ mutants have fewer
TH+ PG cells, although the olfactory
innervation appears to be normal (Qiu et al., 1995; Bulfone et
al., 1998). At this point, it is unclear whether
Dlx2/ and
Dlx5/ mutants have phenotypic
differences in the production of local circuit neurons; ongoing work is
aimed at elucidating the individual and combined roles of the
Dlx genes in OB neurogenesis.
Dlx and GAD expression in the
embryonic brain suggests pathways of olfactory bulb local circuit
neuron migration
On the basis of the expression of Dlx1,
Dlx2, Dlx5, and GAD67, there may be at
least three distinct progenitor zones that contribute tangentially
migrating precursors to OB local circuit neurons (Fig.
5A,C; and data not shown). Cells
may migrate from the SVZ of the septum to medial parts of the OB (Fig.
5C), in addition to cells that may migrate from the SVZ of
the MGE and LGE into lateral parts of the OB (Fig. 5A). In
each case, these progenitors enter the ventral part of the OB (Fig. 5).
We propose that the rostral convergence of the three embryonic pathways
becomes the RMS. The number of progenitor zones and potential migration pathways suggests that different subtypes of OB local circuit neurons
may originate from distinct locations.
However, DLX5 function is not essential for the formation of these
progenitor zones or potential migration pathways (Fig. 5B,D,H,J).
Indeed, in E18.5 Dlx5/ mutants,
tangential migration along the RMS was detectable using the retroviral
and DiI-labeling assays (Fig. 8E,F;
and data not shown). We generally detected decreases in migration;
however, because our assays were not quantitative, it is premature to
conclude that DLX5 is essential for normal levels of migration in the
RMS. Furthermore, we did not detect gross abnormalities in the
proliferation of OB local circuit neuron progenitors in the
Dlx5/ mutants (Fig. 7; and data not
shown). However, subtle defects in proliferation would not have been
detected with our methods.
Thus, why do Dlx5/ mutants have fewer
GAD65+,
GAD67+, and
TH+ neurons in the GC and PG
(Figs. 4, 5, 9)? A BrdU pulse chase (E16.5-E18.5) shows a reduction in
the number of cells that populate the OB mantle zone (Fig.
8C,D). This suggests that the reduction in OB local circuit neurons is attributable to their reduced production. If
the defect were attributable to reduced migration of postmitotic neurons, we would have observed periventricular ectopic collections of
postmitotic neurons. Unlike the
Dlx1&2/ mutants, in which this is a
prominent aspect of their phenotype in the basal ganglia (Anderson et
al., 1997; Marin et al., 2000), periventricular ectopic neurons were
not found in the Dlx5/ mutants.
Therefore, we suggest that DLX5 is necessary for progenitors in the SVZ
of the OB to mature into postmitotic local circuit neurons. This defect
could cause an expansion of the progenitor zone (Fig. 9). This would be
consistent with the apparent accumulation of BrdU-labeled cells in the
SVZ in the BrdU pulse chase (E14.5-E18.5) (Fig.
8A,B). Furthermore, although there
is not a massive expansion of the SVZ, this defect could explain why
the olfactory ventricle is smaller in most mutants (Fig. 2, 4, 6-8).
Thus, future studies will focus on establishing how DLX5 regulates the
transition from SVZ-type progenitor to neuron.
TOP
ABSTRACT
Introduction
