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The Journal of Neuroscience, March 1, 2003, 23(5):1769
Widespread Defects in the Primary Olfactory Pathway Caused by
Loss of Mash1 Function
Richard C.
Murray1, 3,
Daniel
Navi1, 3,
John
Fesenko2, 3,
Arthur D.
Lander2, 3, and
Anne L.
Calof1, 3
1 Department of Anatomy and Neurobiology,
2 Department of Developmental and Cell Biology, and
3 Developmental Biology Center, University of California,
Irvine, Irvine, California 92697-2300
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ABSTRACT |
MASH1, a basic helix-loop-helix transcription factor, is widely
expressed by neuronal progenitors in the CNS and PNS, suggesting that
it plays a role in the development of many neural regions. However, in
mice lacking a functional Mash1 gene, major alterations have been reported in only a few neuronal populations; among these is a
generalized loss of olfactory receptor neurons of the olfactory epithelium. Here, we use a transgenic reporter mouse line, in which the
cell bodies and growing axons of subsets of central and peripheral
neurons are marked by expression of a tau-lacZ reporter
gene (the Tattler-4 allele), to look both more broadly and deeply at defects in the nervous system of Mash1 /
mice. In addition to the expected lack of olfactory receptor
neurons in the main olfactory epithelium, developing
Mash1/;Tattler-4+/ mice exhibited reductions in
neuronal cell number in the vomeronasal organ and in the olfactory
bulb; the morphology of the rostral migratory stream, which gives rise
to olfactory bulb interneurons, was also abnormal. Further examination
of cell proliferation, cell death, and cell type-specific markers in
Mash1/ animals uncovered parallels between the main
olfactory epithelium and the vomeronasal organ in the regulation of
sensory neuron development. Interestingly, this analysis also revealed
that, in the olfactory epithelium of Mash1/ animals,
there is an overproduction of proliferating cells that co-express
markers of both neuronal progenitors and supporting cells. This finding
suggests that olfactory receptor neurons and olfactory epithelium
supporting cells may share a common progenitor, and that expression of
Mash1 may be an important factor in determining whether
these progenitors ultimately generate neurons or glia.
Key words:
bHLH transcription factor; olfactory epithelium; olfactory receptor neuron; transgenic mouse; olfactory bulb; rostral
migratory stream; subventricular zone; vomeronasal organ; neural
progenitor; lineage; supporting cell; granule cell
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Introduction |
Basic helix-loop-helix (bHLH)
transcription factors appear to play a conserved role in determining
neuronal fate during development (Brunet and Ghysen, 1999 ; Guillemot,
1999). In Drosophila, proneural genes such as
achaete, scute, and atonal instruct
neuronal fate determination (Jan and Jan, 1994), and at least some
homologs of these genes play analogous roles in vertebrates. For
example, loss of function studies in mice have shown that
Mash1, Ngn1, and Ngn2 are required for
the development of specific subsets of neurons (Guillemot et al., 1993;
Fode et al., 1998; Ma et al., 1998).
Mash1, a homolog of achaete and scute,
is expressed by neuronal progenitors in the developing PNS and CNS. In
the CNS, Mash1 is expressed in the developing telencephalon,
including ventricular zone (VZ) of the developing olfactory bulb (OB)
and ganglionic eminences, diencephalon, midbrain, spinal cord, and
retina (Guillemot and Joyner, 1993; Ma et al., 1997; Horton et al.,
1999). Mash1 is also expressed in the PNS, including the
olfactory epithelium (OE) and the sympathetic, parasympathetic, and
enteric nervous systems (Guillemot and Joyner, 1993; Gordon et al.,
1995; Blaugrund et al., 1996; Ma et al., 1997). Although this
widespread expression suggests that Mash1 plays a role in
the development of many neural regions, the initial analysis of
gene-targeted Mash1/ mice detected deficits only in
olfactory receptor neurons (ORNs) and neurons of the autonomic nervous
system (Guillemot et al., 1993). Subsequent studies demonstrated
additional and more subtle roles for Mash1, often acting in
concert with other transcriptional regulators, in regulating cell fate
and neuronal differentiation in various CNS regions (Casarosa et al.,
1999; Horton et al., 1999; Torii et al., 1999; Tomita et al., 2000;
Hatakeyama et al., 2001; Marquardt et al., 2001; Nieto et al., 2001).
These findings, and the discovery of thalamocortical axon pathfinding
defects resulting from loss of Mash1 (Tuttle et al., 1999),
suggest that other neural deficits are likely to be present in
Mash1/ animals.
To look systematically for additional phenotypes in Mash1/
animals, as well as other neurodevelopmental mutants, we developed a transgenic mouse line (Tattler-4) that allows rapid
visualization of subsets of neurons and their axons during development.
Tattler-4 mice were generated using a promoter fragment from the
T 1 -tubulin gene to express a
tau-lacZ fusion gene. The transgene is expressed in cell
bodies and axons of subsets of PNS and CNS neurons during terminal
neuronal differentiation and initial axon outgrowth. By breeding the
Mash1 null allele onto Tattler-4 and examining the primary
olfactory pathway with  -galactosidase histochemistry, we were able
to identify abnormalities in Mash1/ animals not only in
OE, but also in the vomeronasal organ (VNO), OB, and rostral migratory
stream (RMS), a structure containing neural progenitors that give rise
to OB interneurons. Analysis of gene expression in the developing OE
and VNO of Mash1/ animals uncovered parallels in genetic
regulation of sensory neuron development between these two tissues and
provided clues that ORNs and OE supporting cells share a common progenitor.
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Materials and Methods |
Transgenic mice. The
T1:tau-lacZ transgene was generated
by ligating a 5.5 kb XhoI/SpeI fragment
containing the tau-lacZ fusion and SV40 splice site and
poly(A) addition sequence (Callahan and Thomas, 1994) downstream of a
1.1 kb BssHII/XbaI fragment of the
T1 -tubulin promoter (Gloster et
al., 1994) (see Fig. 1A). To generate transgenic
mice, the T1:tau-lacZ construct was linearized with AscI/PmeI and injected into
pronuclei of fertilized mouse ova (CB6 F2) using standard techniques
(Hogan, 1994) in the University of California, Irvine, Transgenic Mouse
Facility. Transgenic offspring were identified by PCR of tail DNA and
bred against CD-1 mice (Charles River Laboratories,
Wilmington, MA) to generate four independent transgenic mouse lines,
named T1-tubulin tau-lacZ expressing reporter (Tattler) 1-4.
Tattler-4 mice, used in this study, were bred for more than eight
generations on a CD-1 background. Mash1+/- mice were a
generous gift from F. Guillemot (IGBMC, Strasbourg, France) (Guillemot
et al., 1993) and were maintained on a CD-1 background, where the OE
phenotype is fully penetrant (Cau et al., 1997; this study).
Tattler-4+/+;Mash1+/- animals were mated with
Mash1+/- females to generate
Tattler-4+/-;Mash1+/+ and Tattler-4+/-;Mash1/
littermate embryos for analysis.
Genotype was determined by PCR (30 cycles, annealing temperature
58°C) of tail or yolk sac DNA using oligonucelotide primers (Invitrogen, San Diego, CA) specific for lacZ
for Tattler-4 animals or for Mash1 and neo to
distinguish Mash1+/+, +/, /
genotypes (lacZ, GenBank accession number V00296;
LacZ-Forward, 5'-TGATGAAAGCTGGCTACAT-3'; LacZ-Reverse,
5'-ACCACCGCACGATAGAGATT-3'; Mash1, GenBank U68534; Mash1-Forward, 5'-CCAACTGGTTCTGAGGAC-3'; Mash1-Reverse,
5'-CCCATTTGACGTAGTTGG-3'; neo, GenBank U43611; Neo-Forward,
5'-GATCTCCTGTCATCTCACCT-3'; Neo-Reverse,
5'-ATGGGTCACGACGAGATCCT-3').
In situ hybridization, immunohistochemistry, and terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining. One hour before they were killed,
timed-pregnant female mice were injected intraperitoneally with 50 µg/gm body weight of 5-bromo-2'-deoxyuridine (BrdU) (5 mg/ml in 0.9%
saline, 0.007N NaOH). Pregnant dams were killed, and embryos were
dissected and fixed overnight in 4% paraformaldehyde in 0.02 M NaPO4, 0.15 M NaCl, pH 7.5. Embryos were washed in PBS,
cryoprotected in 30% sucrose/PBS, and sectioned at 12 or 20 µM on a cryostat. Sections were collected on
Superfrost/Plus slides (Fisher Scientific, Houston, TX)
and stored at 80°C until use. For staging of embryos, 12:00 P.M. on
the day a vaginal plug was detected was designated embryonic day (E)
0.5.
For in situ hybridization, sections were fixed onto slides
in 4% paraformaldehyde/PBS, washed in PBS, incubated with proteinase K
(25 µg/ml, 10-15 min), refixed in 4% paraformaldehyde/PBS,
acetylated (0.25% acetic anhydride in 0.1 M
triethanolamine, pH 8.0), and hybridized at 60°C in 50% formamide,
5× SSC, 300 µg/ml Yeast tRNA, 100 µg/ml heparin, 1× Denhardt's,
0.1% Tween 20, 0.1% CHAPS, and 5 mM EDTA
containing 100 ng/ml probe. Unbound probe was removed by washing (0.2×
SSC, 60°C), and probes were detected using alkaline phosphatase-conjugated sheep anti-DIG antibodies (1:2000)
(Roche Molecular Biochemicals, Indianapolis, IN) and
visualized using 5-bromo-4-chloro-3-indolyl-phosphate
(BCIP)/4-nitroblue tetrazolium chloride (NBT) as substrate. Slides were
dehydrated and mounted in Pro-Texx (Lerner Laboratories,
Pittsburgh, PA) before viewing. The probes that were used were as
follows: 375 bp fragment of mouse Mash1 coding region, 2.0 kb fragment of mouse Mash1 gene including coding region and
3'UTR [Clone 1 in Guillemot and Joyner (1993)], 1.2 kb fragment of
rat ngn1 gene (Ma et al., 1996), 391 bp fragment of mouse
Ncam coding region (Barthels et al., 1987), 1.7 kb fragment
of the mouse Trp2 coding region (Vannier et al., 1999), and
155 bp fragment of the mouse reelin coding region
(D'Arcangelo et al., 1995). The steel probe consisted of an
879 bp fragment of the mouse steel coding region and 3'UTR
(GenBank M57647) (bp 386-1265), generated by RT-PCR of mouse E12.5
head total RNA. The PCR product was cloned into the pCR2.1 vector
(Invitrogen), and its identity was confirmed by
sequencing. The Gad67 probe consisted of an 867 bp fragment
of the mouse Gad67 3'UTR (GenBank NM008077) (bp 1987-2854),
generated by RT-PCR of mouse P1 brain total RNA. The PCR product was
cloned into pBluescript (Stratagene, La Jolla, CA), and
its identity was confirmed by sequencing.
For BrdU immunostaining, sections were permeabilized in 0.1%
Triton X-100 in PBS (30 min, room temperature), treated
with 2N HCl (1 hr, 37°C), neutralized in HBSS
(Invitrogen), blocked in 0.1% Triton X-100,
10% bovine calf serum (BCS) (HyClone, Logan, UT) in PBS,
incubated with anti-BrdU antibody (clone BU1/75) (1:1000 in 10% BCS in
PBS; overnight, 4°C) (Harlan Sera-Lab,
Sussex, UK), and visualized with Texas Red-conjugated goat anti-rat IgG
(1:50) (Jackson ImmunoResearch, West Grove, PA).
Immunostaining with monoclonal anti-neuron-specific nuclear protein
(NeuN) (1:500 dilution) (Chemicon, Temecula, CA) was
performed in the same manner but without acid treatment, and primary
antibody was detected using Texas Red-conjugated goat anti-mouse IgG1
(1:50) (Southern Biotechnology, Birmingham, AL). For
immunostaining with 12F8 monoclonal anti-polysialic acid (PSA) neural
cell adhesion molecule (NCAM) (Carl Lagenaur, University of
Pittsburgh), hybridoma supernatant was applied overnight at 4°C and
detected using Texas Red-conjugated goat anti-rat IgM (1:50)
(Jackson ImmunoResearch).
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling (TUNEL) staining to detect DNA fragmentation in
situ was performed as described previously (Holcomb et al., 1995),
using Texas Red-conjugated NeutrAvidin (Molecular Probes, Eugene, OR) to detect incorporated Biotin-16-dUTP (Roche
Molecular Biochemicals).
-galactosidase histochemistry. E13.5 whole-mount staining
was performed as described previously (Murray et al., 2000). For older
embryos, tissues were dissected and fixed by immersion in 2 mM MgCl2, 4%
paraformaldehyde in 0.02 M
NaPO4, 0.15 M NaCl, pH 7.5, for 2-4 hr at room temperature, washed three times 5 min in 2 mM MgCl2 in PBS at room
temperature, cryoprotected in 30% sucrose, 2 mM
MgCl2 in PBS, and sectioned at 30 µm on a
cryostat. Sections were collected on gelatin-coated slides, postfixed
in 2 mM MgCl2, 0.5%
glutaraldehyde, in PBS for 15 min, and permeabilized in 2 mM MgCl2, 0.1%
Triton X-100, 0.01% deoxycholate, in PBS for 10 min.
Sections were stained overnight in 1 mg/ml 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-gal), 5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6, 2 mM MgCl2, 0.1%
Triton X-100, 0.01% deoxycholate, in PBS at 37°C, and
then dehydrated and mounted in Pro-Texx.
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Results |
Subsets of neurons in the PNS and CNS are marked in Tattler-4
transgenic mice
To study the connectivity of developing neuronal populations in
the embryonic mouse nervous system, we generated transgenic mice that
express an axon-targeted reporter gene in differentiating neurons. A
1.1 kb fragment of 5' regulatory sequence from the T1 tubulin gene was used to drive expression
of a reporter construct (tau-lacZ), which consisted of a
fragment of the bovine gene encoding the microtubule-associated
protein, tau, fused to the bacterial -galactosidase gene
(lacZ) (Callahan and Thomas, 1994; Gloster et al., 1994).
The resulting construct (T1:tau-lacZ) (Fig.
1A) was used to
generate four independent transgenic mouse lines, which were named
T1-tubulin tau-lacZ expressing reporter (Tattler) 1-4.
The Tattler-4 strain had the most extensive pattern of expression and
was chosen for further analysis.
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Figure 1.
A, Diagram of the
T1:tau-lacZ transgenic construct used
to generate Tattler-4 mice. The tau-lacZ reporter fusion
is expressed under the control of the
T1 -tubulin
promoter. A polyadenylation signal from SV40 is included in the
construct. A, AscI; X,
XhoI; P, PmeI.
B-I, T1:tau-lacZ
expression in Tattler-4 transgenic mice. Expression was visualized by
X-gal staining, as detailed in Materials and Methods. B,
Sagittal view of E13.5 Tattler-4 transgenic embryo stained with X-gal.
Staining is present in the neural retina (NR), olfactory
epithelium (OE), olfactory bulb (OB),
tectum (T), cerebellum (Cb),
dorsal spinal cord (SC), trigeminal ganglion
(Vthg), and dorsal root
ganglia (DRG). Sensory nerves in the limb
(arrow) are also stained. C, Dorsal view
of E13.5 embryo. The sympathetic chain (Sym) underlying
the spinal cord, dorsal root ganglia (DRG), cutaneous
(Cut), and intercostal (In) spinal nerves
are stained. D, Transverse section through the spinal
cord of an E13.5 embryo. Staining is present in the dorsal spinal cord
(SC) and dorsal root ganglia (DRG) as
well as in the sympathetic chain (Sym). Staining in the
ventral root (VR) likely represents preganglionic
sympathetic axons, because the ventral horn motor neurons are not
stained. The dorsal aorta (DA) is labeled for reference
(dorsal is at the right). E, Horizontal
section through the abdomen of an E13.5 embryo. Staining is present in
the enteric plexus (Epl) of the developing gut.
F, Horizontal section through the head of an E13.5
embryo stained in whole mount. Staining is present at the margin of the
cerebral cortex (C), pons
(P), cerebellum (Cb), diencephalon
(Di), ganglionic eminence (GE), and
hippocampus (Hi). The oculomotor
(IIIrdn) and trochlear
(IVthn) cranial nerves
are also stained. G, Horizontal section through the eye
of an E16.5 embryo. Prominent staining is present in the retinal
ganglion cells (RGC) and their axons in the optic nerve
(IIndn). Some staining
in the developing outer nuclear layer (ONL) is also
apparent. H, Parasagittal section through the head of a
P0 animal. Staining is present in the OE,
OB, intermediate zone of the cerebral cortex
(Ciz), hippocampus (Hi), fimbria
(Fim), thalamus (Th), optic chiasm
(OC), hypothalamus (Hy), tectum
(T), inferior colliculus (IC), cerebellum
(Cb), and trigeminal ganglion
(Vthg).
I, Parasagittal section through the cerebellum of a P0
Tattler-4 mouse. Staining is present in cells of the external granule
layer (EGL). Scale bars: D,
E, G, I, 100 µm;
F, 300 µm; H, 1 mm.
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To reveal the overall expression pattern of
T1:tau-lacZ expression in Tattler-4 mice,
transgenic embryos were fixed and stained with X-gal as whole mounts at
day 13.5 of gestation (E13.5). As shown in Figure 1, B and
C, X-gal staining was specific to neurons and was present in
both neuronal cell bodies and axons in a subset of neural structures in
both the CNS and PNS. In the CNS, prominent staining was observed in
the retina, olfactory bulbs, telencephalon, diencephalon, midbrain,
hindbrain, and spinal cord (Fig. 1B). In addition,
cranial sensory ganglia and nerves, including the trigeminal,
oculomotor, and trochlear nerves, were labeled by X-gal staining (Fig.
1B). In the PNS, expression was seen in the dorsal
root ganglia and cutaneous and intercostal spinal nerves (Fig.
1C). Sections made from E13.5 X-gal-stained whole mounts revealed that both sympathetic nerve fibers (Fig. 1D)
and the enteric plexus were labeled (Fig. 1E). These
sections also revealed that staining of the dorsal spinal cord could be
attributed to labeled fibers of the dorsal root entry zone; developing
motoneurons and interneurons were not labeled (Fig.
1D). Brain sections from E13.5 whole mounts showed
X-gal staining at the margins of structures such as cortex,
diencephalon, ganglionic eminence, and pons, but not in the respective
ventricular zones (Fig. 1F). This suggests that the
transgene is expressed primarily by postmitotic neurons rather than
neuronal progenitors and is consistent with what is known about time of
onset of terminal neuronal differentiation in these regions (Angevine,
1970; Pierce, 1973; Nornes and Carry, 1978; McConnell, 1981). Because
previous studies have shown that the T1
tubulin promoter fragment that we used drives reporter gene
expression in embryonic neurons at the time of terminal differentiation and initial axon outgrowth (Gloster et al., 1994, 1999), these observations suggested that X-gal staining in Tattler-4 mice reveals the axons and, to a lesser extent, cell bodies, of newly
differentiating neurons.
Examination of Tattler-4 embryos at older ages revealed high levels of
reporter expression in distinct subsets of neurons in different regions
of the CNS. For example, in the developing neural retina at E16.5,
retinal ganglion cells and their axons were darkly stained; light
staining was also observed in cells of the developing outer nuclear
layer (ONL) (Fig. 1G). At postnatal day (P) 0, staining
could be seen in a subset of cells and their axons in the cerebral
cortex, hippocampus, fimbria, and striatum; fiber tracts in the
diencephalon and midbrain were also labeled (Fig.
1H). Especially prominent staining was observed in
the ORNs of the OE and associated olfactory nerve, the glomeruli and
mitral/tufted cell layer of the olfactory bulb; and cells of the
external granule layer, but not Purkinje cells, in the cerebellum (Fig.
1H,I). Expression of the
T1:tau-lacZ transgene in only a subset of CNS and PNS neurons can likely be attributed to sensitivity of the T1-tubulin promoter fragment to the site of
transgene integration; other investigators have generated transgenic
lines using this promoter to drive a nuclear lacZ reporter
gene, and the subset of expressing neurons varied in different lines
(Gloster et al., 1994). Because expression of
T1:tau-lacZ by specific populations of neurons
in Tattler-4 mice is a stable characteristic of all mice in this
transgenic line, it provides a useful tool for selectively examining
the behavior of these cells as development of the nervous system proceeds.
T1:tau-lacZ reporter gene
expression reveals unexpected changes in the olfactory bulb and rostral
migratory stream of Mash1/ mice
Because the T1:tau-lacZ reporter gene is
expressed strongly in several structures of the primary olfactory
pathway in Tattler-4 mice, we used these animals to help us examine
development of this pathway in animals in which the gene encoding the
basic helix-loop-helix transcription factor, MASH1, has been disrupted
by homologous recombination. Mash1/ mice die at birth
and have been demonstrated previously to have a profound reduction in
the number of ORNs in the OE lining the nasal cavity (Guillemot et al.,
1993). Because Mash1 is known to be expressed in the
developing telencephalon, including the OB (Guillemot and Joyner, 1993;
Sommer et al., 1996), we hypothesized that other defects might be
present in the primary olfactory pathway of Mash1/ mice.
To test this possibility, we bred the Mash1 knock-out
allele onto the Tattler-4 background and compared olfactory
structures in Mash1/ animals and their wild-type littermates.
We first examined mice around the time of birth (E18.5/P0), because
Mash1/ animals only survive to this age. At E18.5,
X-gal staining of Mash1+/+;Tattler-4+/ mice revealed
expression of tau-lacZ in ORN cell bodies within the OE and
in ORN axons projecting to the OB (Fig.
2A). Also labeled by
X-gal staining were cells in the mitral/tufted cell layer of the OB;
these are the cell types onto which ORNs synapse. However, cells in the
developing OB granular layer, which contains primarily granule cell
interneurons, were not stained (Fig. 2A) (Farbman,
1992). In contrast to the situation in wild-type embryos, the OE of
Mash1/;Tattler-4+/ embryos was devoid of X-gal
staining and was much thinner than normal, and ORN axons were virtually
absent (Fig. 2B). Thus, the Tattler-4
allele clearly revealed the deficit in ORN development known to occur
in the absence of Mash1 function.
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Figure 2.
Alterations in the OE and OB of Mash1/
mice. A, B, E18.5 littermate
embryos heterozygous for the Tattler-4 reporter allele
and Mash1+/+ (A) or
Mash1/ (B) were sectioned at
30 µm in the sagittal plane and stained with X-gal. Anterior is on
the left; dorsal is at the top. The
olfactory epithelium (OE), mitral/tufted cell layer
(MC), outer nerve layer (NL), olfactory
nerve (Istn), and
granular layer (asterisk) are shown. Scale bar, 300 µm. C, Diagram to illustrate the measurements taken to
quantify changes in OB and AOB size. OB diameter and granular layer
(GL) thickness were measured in the plane perpendicular
to the cribriform plate, at the point midway between the anterior tip
of the OB and the flexure at the junction of the anterior border of the
cerebral cortex and the dorsal surface of the OB (labeled
brackets). OB diameter was measured as the distance between the
dorsal and ventral surfaces of the OB; GL thickness was measured as the
distance across the unstained granular layer in X-gal-stained tissue.
AOB area (light blue shading) was measured using NIH
Image 1.61. D, Decreased OB diameter in
Mash1/ embryos. To quantify changes in the size of
the OB, serial 30-µm-thick sagittal sections through the entire
extent of both OBs were measured in two Mash1/;Tattler-4+/
and two Mash1+/+;Tattler-4+/ animals at E18.5.
A total of 67-80 measurements were taken per animal and plotted
relative to the distance of the measured section from the midline.
Mash1/ embryos (blue lines) and
wild-type embryos (red lines) are shown.
E, Decreased GL thickness in Mash1/
embryos. A total of 32-50 measurements were taken per animal
and plotted as in D. F, Reduced AOB area
in Mash1/ embryos. A total of 24-35 measurements
were taken per animal and plotted as in D.
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X-gal staining of Mash1/;Tattler-4+/ embryos also
revealed that the OBs of these animals were reduced in size compared with their wild-type littermates (Fig. 2B). In
addition to a size reduction caused by absence of the olfactory nerve
layer of the bulb (expected because of the lack of ingrowing ORN
axons), the granular layer of the OB appeared to be greatly reduced in
size in Mash1/;Tattler-4+/ embryos relative to
Mash1+/+;Tattler-4+/ littermates (Fig.
2A,B, asterisks).
Moreover, the accessory olfactory bulb (AOB) (the synaptic target of
vomeronasal sensory neurons) (Halpern, 1987) also appeared to be
smaller in Mash1/;Tattler-4+/ embryos.
To quantify these changes, we measured the diameter of the OB, the
thickness of the granular layer (the cell layer containing developing
granule interneurons) within the OB, and the area of the AOB
through the entire extent of both bulbs in two
Mash1/;Tattler-4+/ and two
Mash1+/+;Tattler-4+/ littermate embryos (Fig.
2C). The data, shown in Figure 2D-F,
demonstrate clear reductions in size of both the OB and the AOB in
Mash1 mutant animals. The diameter of the OB of
Mash1/;Tattler-4+/ animals was reduced by 27% relative to their Mash1+/+;Tattler-4+/ littermates, and
the thickness of the granular layer was reduced by 45% in
Mash1 mutants relative to wild-type littermates. The average
area of the AOB also showed a large reduction, being decreased by 30%
in Mash1/;Tattler-4+/ animals relative to their
Mash1+/+;Tattler-4+/ littermates. To confirm that the
reduction in the size of the OB in Mash1/;Tattler-4+/ animals is not caused by the Tattler-4 allele,
the diameter of the OB was measured in Mash1/ animals
and their wild-type littermates maintained on a CD-1 background. A
similar decrease in OB diameter (29%) in Mash1/ animals
relative to wild types was observed (wild type, 0.91 ± 0.03 mm;
Mash1/, 0.65 ± 0.01 mm, for three animals of each
genotype; more than eight sections measured per animal).
Granule cell interneurons of the OB originate in the subventricular
zone of the lateral ventricles and migrate to the OB in a structure
known as the RMS (Luskin, 1993; Lois and Alvarez-Buylla, 1994).
The Tattler-4 reporter allele is expressed by cells of the
RMS, allowing this structure to be visualized easily in X-gal-stained sections of normal and Mash1/ animals on the
Tattler-4 background. In sagittal sections through the
brains of wild-type animals at E18.5, the RMS could be seen as a stream
of cells, many of which were stained with X-gal, extending from the
anterior limit of the lateral ventricle to the caudal boundary of the
OB (Fig. 3A). Interestingly,
in Mash1/;Tattler-4+/ embryos, the RMS was present, but its shape was different, being noticeably thicker in
Mash1/;Tattler-4+/ animals than in their wild-type
littermates (Fig. 3B). Moreover, the density of
X-gal-stained cells in the RMS of Mash1/;Tattler-4+/ animals was consistently greater than what was observed in wild types (Fig. 3, compare C, D). Because induced
absence of the highly sialylated form of NCAM is associated with
similar morphological changes in the RMS, as well as reduced OB size
(Bruses and Rutishauser, 2001), we used a monoclonal antibody specific
for the PSA moieties on the "embryonic" form of NCAM to stain
sections through the RMS in E17.5 Mash1/ and
Mash1+/+ littermates (Chung et al., 1991). As shown in
Figure 3E, in wild-type embryos, essentially the entire
forebrain and OB are stained with the 12F8 monoclonal anti-PSA-NCAM.
The cell-dense RMS and ventricular zone of the OB (visualized with a
nuclear stain in Fig. 3G) are also immunopositive for
PSA-NCAM, although as described previously, staining is less intense
than in the surrounding brain regions (Chung et al., 1991). In
Mash1/ embryos, the level of anti-PSA-NCAM
immunoreactivity observed in the RMS and OB ventricular zone was
similar to that in wild types (Fig.
3F,H). Thus, there is no
obvious change in expression of PSA-NCAM in the absence of
Mash1 function.
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Figure 3.
Comparison of the rostral migratory stream in
Mash1/ and Mash1+/+ embryos.
A-D, Thirty micrometer sagittal sections through the
head of an E18.5 Mash1+/+;Tattler-4+/ embryo
(A, C) and a
Mash1/;Tattler-4+/ littermate (B,
D) stained with X-gal. C,
D, Higher power images of the regions
bracketed in A and B.
E-H, Twelve micrometer sagittal sections through the
head of an E17.5 Mash1+/+ embryo (E,
G) and a Mash1/ littermate
(F, H) processed for PSA-NCAM
immunostaining (monoclonal anti-PSA-NCAM 12F8) (Chung et al., 1991)
(E, F) or nuclear DNA staining
(bisbenzimide H 33258 counterstain of same sections) (G,
H). Inset in F
shows a negative control (no primary antibody) section of forebrain,
including a portion of the lateral ventricle, for comparison. Rostral
migratory stream (RMS) and ventricular zone
(VZ) are shown. Scale bars: (in B)
A, B, 250 µm; (in D)
C, D, 50 µm; (in
H) E-H, 300 µm.
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Many of the neuronal cells migrating in the RMS are known to be
progenitors of OB granule cells; these progenitors are unusual in that
they express markers characteristic of differentiated neurons (e.g.,
neuronal tubulins) while still continuing to proliferate (Luskin,
1998). Granule cell progenitors follow a stereotyped migratory route
into the OB: they first migrate tangentially into the OB along the
ventricular zone and then disperse radially into the surrounding
granular layer (Alvarez-Buylla, 1997; Luskin, 1998). To determine
whether this pattern of cell migration is disrupted in Mash1/
animals, E17.5 embryos were given a pulse of BrdU to label
migratory progenitors and then killed 1 hr later, and their brains were
fixed and processed for BrdU immunoreactivity. As shown in Figure
4, A and B, the
patterns of dispersal of BrdU-labeled cells were very different in
Mash1/ embryos and their wild-type littermates. In
wild-type embryos, many BrdU+ cells could be seen outside of the
ventricular zone, dispersed within the granular layer (Fig.
4A). In contrast, BrdU+ cells in the OB of
Mash1/ animals were restricted to the ventricular zone,
which itself appeared thinner than that of wild types (Fig.
4B). These observations were confirmed by counting
the number of BrdU-labeled cells in three concentric bands surrounding
the ventricle of the OB in these sections (Fig. 4C). In
Mash1+/+ embryos, many cells were found to have migrated
radially out of the ventricular zone and into the surrounding granular
layer (i.e., out of Band I into Bands II and III), whereas in
Mash1/ animals, virtually all BrdU-positive cells in the
OB were found within the ventricular zone (Band I). This observation
suggested that, in Mash1/ animals, granule cell
progenitors may fail to migrate out of the RMS and into the developing
granular layer of the OB, resulting in a deficit in differentiated
granule cells and a marked reduction in OB size.
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Figure 4.
Alterations in RMS, VZ, and granular layer of the
OB in Mash1/ embryos. A,
B, D-K, Sagittal sections, 12 µm
(A, B, F,
I) and 20 µm (D,
E, G, H, J,
K), through the heads of E17.5
Mash1+/+ embryos (A, D,
F-H) and Mash1/ littermates
(B, E, I-K) were
processed for BrdU or NeuN immunostaining or in situ
hybridization for 3' Mash1 UTR, Gad67, or
Reelin as indicated. Scale bars: (in B)
A, B, 200 µm; (in
I) F, I, 100 µm;
(in E) D, E,
G, H, J, K,
200 µm. C, Quantification of
BrdU-incorporating cells in the ventricular zone and granular layer of
the OB in Mash1/ embryos (gray
bars) and wild-type (striped bars) littermates.
The total number of BrdU+ cells was counted in a series of three
83-µm-wide (approximate width of the ventricular zone) bands
proceeding dorsally or ventrally from the OB ventricular surface
(inset). Values: Band I, 107 (range, ±11) BrdU+ cells
(wild type) and 85.5 (range, ±10.5) BrdU+ cells
(Mash1/); Band II, 103 (range, ±6) BrdU+ cells
(wild type) and 2 (range, ±2) BrdU+ cells (Mash1/);
Band III, 27 (range, ±2) BrdU+ cells (wild type) and 8.5 (range, +4.5)
BrdU+ cells (Mash1/).
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To investigate this idea, we performed in situ hybridization
experiments to determine whether, in Mash1/ animals, the
BrdU+ cells apparently "trapped" in the RMS and VZ of the OB are
progenitors that would normally express the Mash1 gene. To
do this, we took advantage of the fact that the noncoding second exon
of the Mash1 gene was still present in the targeting vector
used to generate the Mash1/ animals that were used in
our study (Guillemot et al., 1993). It has been shown previously that
transcripts continue to be made from the disrupted Mash1
allele in CNS neural progenitors present in Mash1/
animals (Horton et al., 1999). This finding suggested that a probe
that includes the Mash1 3'UTR could be used to identify
cells that express Mash1 transcripts in Mash1/ animals, as well as normal Mash1-expressing progenitors
in wild types. When we performed in situ hybridization
experiments using such a probe (3' Mash1 probe), we found
that the pattern of hybridization in the RMS/VZ and developing granular
layer of the OB showed close correspondence to the pattern of anti-BrdU
immunoreactivity (Fig. 4D,E). Cells
positive for the 3' Mash1 probe were present in the VZ and
dispersed within the developing OB granular layer in wild-type animals
(Fig. 4D), whereas they were restricted to the VZ in
the OB of Mash1/ animals (Fig. 4E).
This finding suggested that the cells to which the Mash1 3'
probe hybridized are neuronal progenitors that would normally express
Mash1 and migrate out of the RMS/VZ and into the granular
layer of the OB. However, in Mash1/ animals, these cells
are unable to migrate and contribute to OB development, apparently
because of a defect resulting from lack of Mash1 function.
To further investigate the role of Mash1 in the development
of intrinsic OB neuronal cell types, we performed in situ
hybridization experiments using specific markers to compare the
relative sizes of differentiated OB cell populations in
Mash1/ animals and their wild-type littermates.
Differentiated granule cells were detected using a monoclonal antibody
to the neuronal nuclear marker NeuN (Mullen et al., 1992) and a cRNA
probe for Gad67 (Bulfone et al., 1998), whereas OB mitral
cells were identified using a probe for Reelin (D'Arcangelo
et al., 1995). As shown in Figure 4, the number of NeuN+ and
Gad67+ cells was drastically reduced in the granular layer
of Mash1/ OB (Fig.
4I,J) compared with wild
type (Fig. 4F,G), indicating that
differentiated granule cells are greatly reduced in number in
Mash1/ OB. In contrast, there was no apparent decrease
in the number of cells expressing the mitral cell marker
Reelin in Mash1/ OB (Fig. 4, compare H, K).
Together, these data indicate that in Mash1/ animals, OB
granule cell progenitors appear to be generated but are unable to
migrate out of the RMS and VZ into the developing granular layer,
resulting in a dramatic decrease in granule cell number and a marked
reduction in OB size.
Mash1 is required for sensory neuron development in
the vomeronasal organ
Although detection of most odors is mediated by ORNs in the main
OE, pheromone detection is mediated by sensory neurons of the VNO, a
tube-shaped sensory epithelium that lies within the ventral portion of
the nasal septum (Halpern, 1987). Like the main OE, the VNO is derived
from the olfactory placode (Farbman, 1992). Although ORN development in
the main OE is Mash1 dependent (Cau et al., 1997), a recent
report has suggested that genesis of sensory neurons in the VNO does
not require Mash1 (Cau et al., 2002); however, that study
examined only early development (E10.5-12.5). Given that much of the
neurogenesis in the VNO is known to occur late during fetal development
and in the early postnatal period (for review, see Halpern, 1987), it
seemed possible that a requirement for Mash1 function might
not yet be evident in the VNO by E12.5. To resolve this question, we
examined the VNO in Mash1/;Tattler-4+/ embryos around
the time of birth (E18.5). In wild-type
(Mash1+/+;Tattler-4+/) animals, the
T1:tau-lacZ transgene is expressed by VNO
sensory neurons, and the vomeronasal nerve connecting the VNO to the
AOB is heavily labeled by X-gal staining (Fig.
5A). However, in
Mash1/ animals, the epithelium is much thinner than in
wild-type animals, and contains almost no X-gal-stained neurons (Fig.
5B). In addition, the size of the vomeronasal nerve is
reduced dramatically. These results indicate that most of the sensory
neurons of the VNO depend on Mash1 for their proper
development. Moreover, they suggest that the decrease in size of the
AOB in Mash1/ animals, shown in Figure 2, is likely to
be caused, at least in part, by the loss of sensory afferents from the
VNO.
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Figure 5.
Alterations in the VNO of Mash1/
mice. A, B, Sagittal sections
through the VNO of an E18.5 Mash1+/+;Tattler-4+/
embryo (A) and a
Mash1/;Tattler-4+/ littermate
(B) stained with X-gal. Anterior is on the
left, and dorsal is at the top.
A, Sensory neurons in the VNO epithelium
(arrowhead) stain with X-gal as do the vomeronasal
nerves (arrows). B, The number of sensory
neurons and the size of the VNO (arrowhead) as well as
the size of the vomeronasal nerve (arrow) is reduced in
the Mash1/;Tattler-4+/ littermate. Scale bar, 100 µm.
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To determine whether other aspects of VNO sensory neuron development
show similarities to ORN development in the main OE, we examined the
VNO for expression of markers characteristic of different cell stages
in the ORN developmental pathway (Fig.
6). ORN development appears to involve
three stages of proliferating neuronal progenitor cells, all of which
are present in the OE at E14.5-15.5: a neuronal stem cell [defined
functionally, because no definitive marker for it has been identified
(Mumm et al., 1996)] gives rise to neuronal progenitors that express
Mash1 (Fig. 6A) (Gordon et al., 1995).
Mash1-expressing progenitors then give rise to the immediate
neuronal precursors (INPs) of ORNs (Calof and Chikaraishi, 1989). INPs
do not express Mash1, but instead express
Neurogenin1 (Ngn1), another proneural gene
homolog encoding a bHLH transcription factor (Fig.
6B) (Cau et al., 1997; Calof et al., 1998, 2002; Wu
et al., 2003). The progeny of INPs rapidly differentiate into
ORNs and express NCAM (Fig. 6C) (Calof and Chikaraishi,
1989; DeHamer et al., 1994; Calof et al., 1998). In Mash1/
animals, development of ORNs ceases early in this pathway, and as
a consequence expression of the INP- and neuron-specific genes,
Ngn1 and Ncam, is drastically reduced in the OE
(Fig. 6E,F) (Cau et al.,
1997).
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Figure 6.
Gene expression in the OE and VNO of
Mash1/ embryos. A-P, Coronal
sections of E14.5 wild-type OE (A-C) and VNO
(G-I) or Mash1/ littermate OE
(D-F) and VNO (J-L) and
E17.5 wild-type (M, N) or
Mash1/ littermate VNO (O,
P) were processed for in situ
hybridization as described in Materials and Methods. Dorsal is at the
top, and lateral is on the right
(A, arrows). Scale bar: (in
F) A-F, 20 µm;
(in L) G-L, 20 µm; (in P)
M-P, 50 µm.
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In the VNO at E14.5, Mash1 is expressed predominantly by
cells in the basal region, with scattered Mash1+ cells
located more apically in the epithelium (Fig. 6G). This
pattern is similar to the pattern of Mash1 expression in the
main OE (Fig. 6A) and is consistent with what is
known concerning the location of proliferating progenitors in VNO
(Weiler et al., 1999). Ngn1 is also expressed in the basal
progenitor cell region of the VNO (Fig. 6H), and the
neuronal marker Ncam is expressed throughout the sensory
neuron-containing layers (Fig. 6I). To determine
whether this is indicative of a developmental hierarchy of gene
expression in the VNO sensory neuron lineage similar to that of ORNs in
the main OE, we asked whether expression of Ngn1 and
Ncam in the VNO is also dependent on Mash1
function. Ngn1 is essentially absent in the VNO of
Mash1/ embryos at E14.5 (Fig. 6K),
consistent with the idea that Ngn1 expression in the VNO is
Mash1-dependent. In addition, a dramatic reduction in the
number of Ncam-expressing neurons was also apparent in the
VNO of Mash1/ embryos (Fig. 6L). By
E17.5, near the time of birth, only a few scattered sensory neurons
remain in the VNO of Mash1/ animals, as indicated by the
decrease of hybridization for probes to either Ncam or the
VNO neuron-specific channel, Trp2 (Fig.
6M-P) (Liman et al., 1999). Altogether, these
findings demonstrate that Mash1 function is required for the
normal development of sensory neurons in the VNO, as it is in the main
OE. In addition, they suggest that similar hierarchies of gene
expression regulate neuronal differentiation in these two sensory epithelia.
Proliferating neural progenitors are present in the olfactory
epithelium and vomeronasal organ of Mash1/
embryos
The failure of sensory neurons to differentiate in the VNO and OE
in the absence of Mash1 function raises questions about the
fate of neural progenitor cells in these structures. Because the OE and
VNO are thinner and have many fewer neurons in Mash1/ animals (Fig. 6), we hypothesized that progenitor cells might also
be absent. To determine whether this was the case, we injected pregnant
dams (day 14.5 of gestation) with BrdU and fixed embryos 1 hr later to
identify cells in S phase. In wild-type embryos at E14.5, BrdU-labeled
cells are located in the basal and apical layers of the OE and in the
basal two-thirds of the VNO, regions in which proliferating progenitor
cells are known to be located in both of these structures (Fig.
7A,B)
(Smart, 1971; Cuschieri and Bannister, 1975). We also found numerous
BrdU+ cells present in both OE and VNO of E14.5 Mash1/
embryos (Fig. 7C,D). Interestingly, BrdU+
cells in the OE and VNO of Mash1/ embryos are altered in
their relative locations within the epithelia compared with wild types.
Rather than being localized to the basal and apical compartments of the
epithelia, BrdU+ cells are present throughout the apical-basal extent
of the epithelia in Mash1/ OE and VNO. In addition, the
shape of BrdU-incorporating nuclei differs in embryos of the two
genotypes. In Mash1/ animals, the nuclei are larger and
spindle-shaped, rather than round or oval in shape, as they are in
wild-type embryos (Fig. 7C,D).
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Figure 7.
. BrdU incorporation and
Mash1 expression in the OE and VNO of Mash1/
embryos. A-H, E14.5 wild-type
(A, B, E,
F) and Mash1/
(C, D, G,
H) littermates were sectioned coronally and
processed for BrdU immunohistochemistry (A-D) or
in situ hybridization with the probe for the 3'
Mash1 UTR (E-H). Scale bar: (in
C) A, C, E,
G, 20 µm; (in D)
B, D, F, H,
20 µm.
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To determine whether the number of proliferating cells is altered in
Mash1/ OE and VNO, we performed cell counts in six to
eight sections from two animals of each genotype. In wild-type OE,
there were 572 (range, ±85) BrdU+ cells per millimeter versus 376 (range, ±2) BrdU+ cells per millimeter OE in Mash1/
animals. Because the OE is thinner in Mash1/ mice,
we also calculated the density of BrdU+ cells per unit area, and these
numbers were very similar: 80 (range, ±25) BrdU+ cells/10,000
µm2 in wild-type OE versus 81 (range,
±6) BrdU+ cells/10,000 µm2 in
Mash1/ OE. Because the VNO is a circular structure in
cross section, it is not possible to accurately count the number of BrdU+ cells per unit length, so only the density of BrdU+ cells per
unit area was obtained for this structure: 234 (range, ±5) BrdU+
cells/25,000 µm2 in wild-type VNO and
201 (range, ±15) BrdU+ cells/25,000 µm2
in Mash1/ VNO. Thus, the density of proliferating cells
is very similar in both the OE and VNO of wild-type and
Mash1/ animals.
To determine whether the proliferating cells in the OE and VNO of
mutant embryos might be neural progenitors, we used the 3' Mash1
in situ hybridization probe to detect cells expressing mutant
Mash1 transcripts (i.e., presumptive neural progenitors). The pattern of hybridization in the OE and VNO of wild-type embryos observed with this probe was identical to that seen in earlier experiments using a probe containing only the Mash1 coding
region (compare Fig. 7E,F with Fig.
6A,G). In Mash1/
embryos, however, the 3' Mash1 probe hybridized to the
vast majority of cells in OE and VNO, and BrdU-incorporating cells
(Fig. 7, compare G, H with C,
D) were among the expressing cells in both structures. These
findings indicate that the proliferating cells present in mutant OE and
VNO are capable of expressing Mash1, and so in this respect
they have at least one characteristic of sensory neuron progenitors.
However, these cells fail to express normal Mash1 or
Ngn1 transcripts, and for the most part they fail to give
rise to sensory neurons in either the OE or VNO (Fig. 6) (Guillemot et
al., 1993; Gordon et al., 1995; Cau et al., 1997), indicating that they
have lost the ability to give rise to neurons.
3' Mash1-expressing cells in Mash1/
olfactory epithelium express a sustentacular cell marker
What then is the fate of 3' Mash1-expressing
proliferating cells in Mash1/ OE and VNO? To determine
whether these cells have characteristics of other cell types in these
epithelia, we first used a commercial antiserum to keratins to mark
horizontal basal cells (Calof and Chikaraishi, 1989); no difference was
observed in the pattern of staining between wild-type and mutant OE and VNO (data not shown). The Steel gene has been shown to be
expressed by supporting cells (sustentacular cells) of the OE, and a
previous study noted that Steel-expressing cells are still
present in the OE of newborn Mash1/ animals (Guillemot
et al., 1993). We generated a Steel probe by RT-PCR (see
Materials and Methods) and performed in situ hybridization
experiments on the OE and VNO of E14.5 and E17.5 Mash1/
embryos and wild-type littermates. The results are shown in Figure
8. In wild-type OE at both ages,
Steel expression is evident in the apical cytoplasm of
sustentacular cells, which are arrayed in a single layer immediately
above Ncam-expressing ORNs in the epithelium (Fig.
8A,B,G,H).In
Mash1/ OE, in contrast, Steel appears to be
expressed by the majority of cells throughout the basal-apical extent
of the epithelium, whereas almost no Ncam-positive ORNs are
present (Fig.
8D,E,J,K).
(Steel is not expressed in either wild-type or
Mash1/ VNO; data not shown.) Thus, there appeared to be
many more Steel-expressing cells in Mash1/
than in wild-type OE, and the position of these cells overlapped
with those incorporating BrdU and expressing the 3' Mash1
UTR (compare Fig. 7C with Fig. 8E,F,K,L).
To determine the extent of this overlap, we counted the percentage of
cells expressing each marker in Mash1/ OE at E14.5:
84.6% (range, ±3.4%) of cells in the mutant OE express Steel, and 96% (range, ±0.4%) of cells in mutant OE also
label with the 3' Mash1 probe, so it must be the case that
most of the cells in the mutant OE express both markers. Thus, in the
absence of Mash1 function, the OE becomes populated by
proliferating cells that have characteristics of both neuronal
progenitors (expression of the 3' Mash1 UTR) and supporting
(sustentacular) cells (expression of Steel).
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Figure 8.
. Ncam,
Steel, and 3' Mash1 expression in
wild-type and Mash1/ embryos. A-L,
E14.5 (A-F) and E17.5
(G-L) embryos were sectioned in the coronal
plane and processed for in situ hybridization.
Sustentacular cell layer (SUS), olfactory receptor
neuron layer (ORNs), basal lamina (BL),
and lamina propria (LP) are shown. Scale bar: (in
F) A-F, 10 µm;
(in L) G-L, 10 µm.
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Increased apoptosis in olfactory epithelium and vomeronasal organ
of Mash1/ embryos
Despite the high level of proliferation in Mash1/
OE and VNO (Fig. 7), by the time of birth these epithelia are much
thinner than those of wild-type animals (Figs. 2, 5). This observation suggests that the proliferating progenitors present in Mash1/ epithelia do not survive, but instead may be dying at an
abnormally high rate. We and others have observed previously that there
is an increased level of apoptotic death in cells of Mash1/
OE at E13.5-15.5 (Calof et al., 1996b; Cau et al., 1997). To
determine whether cells in Mash1/ VNO also display
abnormal levels of apoptosis, we performed TUNEL assays on
Mash1/ VNO at E14.5 (Holcomb et al., 1995) (OE was also
assessed as a positive control). The results are shown in Figure
9. In wild-type epithelia, almost no
TUNEL+ cells can be observed, whereas numerous TUNEL+ cells are present
in both OE and VNO of Mash1/ animals. Counting the number of TUNEL+ nuclei confirmed the dramatic increase in the number
of apoptotic cells in Mash1/ VNO (e.g., the number of TUNEL+ cells was increased more than fivefold in Mash1/
VNO) (Fig. 9F). Thus, despite being able to
proliferate and being capable of expressing markers of both neuronal
progenitor cells (Mash1) and sustentacular cells
(Steel), many cells in the Mash1/ VNO and OE are unable to survive.
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Figure 9.
Apoptosis in the OE and VNO of Mash1/
embryos. A-D, E14.5 wild-type
(A, C) and Mash1/
(B, D) littermates were sectioned
horizontally at 12 µm and processed for TUNEL labeling as described
previously (Holcomb et al., 1995). Scale bar, 20 µm.
E, The number of TUNEL+ cells per millimeter of OE was
counted in a minimum of 20 fields representing at least 5.2 mm of OE in
one animal of each genotype. Values are 1.8 (±0.53 SEM) TUNEL+ cells
per millimeter OE for wild-type and 119.8 (± 10.87 SEM) TUNEL+ cells
per millimeter OE for Mash1/. F, The
number of TUNEL+ cells per square micrometers of VNO was counted in a
minimum of eight fields representing at least 115,000 µm2 of VNO. Values are 4.64 × 10 4 (±0.75 SEM) TUNEL+ cells per square
micrometer VNO for wild-type and 31.8 × 104
(±7.57) TUNEL+ cells per square micrometer VNO for
Mash1/.
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Discussion |
Loss of Mash1 function leads to abnormalities of
olfactory bulb progenitors and granule cells
One of the more striking effects in the CNS that we observed in
Mash1/ mice was a marked decrease in OB size, evident in both the main OB and the AOB (Fig. 2). Although some reduction in size
of the main OB could be expected from the diminished number of
ingrowing ORN and VNO axons (Figs. 2, 5), our observations clearly show
that much of the reduction occurs in a layer of interneurons, the OB
granule cells (Fig. 2E). These neurons derive from
progenitors that are produced in the subventricular zone of the lateral
ventricle, migrate through the RMS into the VZ of the OB, and finally
disperse into the OB granule layer.
The fact that Mash1 is expressed in the VZ of the OB (Fig.
4) (Guillemot and Joyner, 1993; Sommer et al., 1996), as well as the VZ
and subventricular zone of the ganglionic eminences (Casarosa et al.,
1999; Horton et al., 1999), which contribute cells to the RMS
(Wichterle et al., 1999), suggests that loss of Mash1 function directly affects OB granule neurons and/or their precursors. The alternate possibility that reduced afferent (ORN) input to the OB
in Mash1/ mice indirectly affects granule cell
numberis plausible, given data that naris occlusion in early
postnatal rodents can cause granule cell apoptosis in the ipsilateral
OB (Frazier and Brunjes, 1988; Frazier-Cierpial and Brunjes, 1989; Najbauer and Leon, 1995; Fiske and Brunjes, 2001). However, the time course of deafferentation-induced granule cell apoptosis is likely
to be too slow (~10 d) (Petreanu and Alvarez-Buylla, 2002) to explain
the abnormalities in Mash1/ OB at E18.5, just 3-4 d
after the initial generation of granule neurons (Hinds, 1968).
Interestingly, in Mash1/;Tattler-4+/ animals, the RMS
is noticeably abnormal (Fig. 3A,B),
being both shorter and thicker along the anterior-posterior axis and
exhibiting stronger X-gal staining (i.e., increased density of neuronal
cells) (Fig. 3C,D). These changes are reminiscent
of mice in which the polysialylated form of NCAM has been rendered
nonfunctional by gene inactivation or enzymatic treatment (Cremer et
al., 1994; Ono et al., 1994). In such animals, interference with
neuronal cell migration into the OB causes an accumulation of cells in
the RMS and a reduction in OB size (Bruses and Rutishauser, 2001). If
loss of Mash1 function also results in a migration defect in
the RMS, then the fact that Mash1/ animals exhibit
normal expression of PSA-NCAM (Fig.
3E,F) indicates that the
cause of this defect would have to be different. It has previously been
reported that ventral forebrain neuronal progenitors migrate abnormally
in Mash1/ embryos, and this has been linked to premature
neuronal differentiation (Horton et al., 1999). The possibility that OB
granule neurons also differentiate prematurely is certainly consistent
with the observed increased in X-gal staining in the RMS of
Mash1/;Tattler-4+/ animals, as the
T1:tau-lacZ transgene is selectively turned on
during neuronal differentiation (Fig. 1) (Gloster et al., 1999).
Neurogenesis in the vomeronasal organ: parallels with the
olfactory epithelium
Like the main OE, the VNO derives from the olfactory placode,
maintains a neuroepithelial structure, projects to the OB, and uses a
family of specialized seven-transmembrane odorant receptors to
transduce olfactory signals (Halpern, 1987; Dulac, 2000). Despite these
similarities, Cau et al. (2002) recently reported only a modest
reduction in neuron number in the VNO of Mash1/ animals at E12.5 and concluded that the generation of VNO neurons, unlike those
of the main OE, must depend to a large extent on a factor other than
Mash1. Our observations here support a different view. At
E14.5, we observed a substantially reduced number of neurons in the
Mash1/ VNO, and by E17.5 a profound reduction was
obvious (Fig. 6). These data suggest that although the earliest
neurogenesis in the VNO may be Mash1-independent, most of
the later production of neurons, known to occur late in fetal
development and in the early postnatal period (Halpern, 1987), requires
Mash1 function. Interestingly, in the main OE it is also the
case that the very earliest generated neurons (those born by E9.5) are
relatively Mash1 independent, whereas those produced later
require Mash1 (Cau et al., 1997). Thus, our results suggest
that the molecular details of neurogenesis in the VNO and OE may be
more similar than suspected previously. Such a view is supported
further by our finding in the E14.5 VNO that Mash1 is
required for expression not just of neuron-specific markers
(Ncam, Trp2), but also of Ngn1, a gene
that, in the main OE, marks a progenitor cell stage interposed between
Mash1+ cells and neurons (Cau et al., 1997; Calof et al.,
2002).
Do supporting cells and sensory neurons of the olfactory epithelium
share a common progenitor?
Despite the deficit in neuron number in the OE and VNO of
Mash1/ embryos as early as E14.5 (Fig. 6), we found that
many cells in these tissues are proliferating and most express the Mash1 3' UTR (Fig. 7). The observation that the
Mash1 promoter is active in so many more cells in
Mash1/ OE and VNO than in wild types suggests that far
fewer cells in developing OE and VNO normally express Mash1
transcripts than are competent to do so. It also supports the findings
of Horton and colleagues (1999), whose studies of gene expression in
the ventral forebrain of Mash1/ embryos implied that
MASH1 negatively regulates its own expression (Horton et al., 1999).
Those authors argued that, in Mash1 nulls, absence of MASH1
protein (required for Notch-Delta signaling) results in a breakdown of
the lateral inhibition that is necessary for correct specification of
neuronal progenitors from a larger field of competent cells (Lewis,
1996). The idea that Mash1 functions in the OE through a
Notch signaling pathway is supported by the finding that activation of
the expression of several genes in this pathway fails to occur in
Mash1/ OE (Cau et al., 2000, 2002). Altogether, these
observations support a model in which the OE and VNO in
Mash1/ embryos are populated by early proliferating progenitor cells that transcribe (aberrant) Mash1
transcripts, but lacking Mash1 function, fail to
differentiate properly and subsequently undergo apoptosis (Fig. 9).
Interestingly, most (perhaps all) of these presumed early progenitors
in Mash1/ OE express Steel, a marker of
supporting (sustentacular) cells (Fig. 8), together with aberrant
Mash1 transcripts (Figs. 7, 8). This suggests that most of
these cells are developing along a sustentacular cell pathway. They
also exhibit the elongated nuclei characteristic of sustentacular cells
(Fig. 7) (Smart, 1971; Cuschieri and Bannister, 1975). The obvious
implication is that, early in embryonic development, the OE may contain
bipotential progenitors that subsequently become restricted to a
neuronal (ORN) or glial (sustentacular) fate, and that Mash1
function is required for the neuronal determination event. Indeed, in
the inner ear, another placode-derived sensory epithelium, it has been
shown that sensory and supporting cells share a common progenitor (Corwin and Cotanche, 1988; Ryals and Rubel, 1988; Fekete et al., 1998). Moreover, in other areas of the nervous system, bHLH
transcription factors have been shown to act to promote neuronal, and
inhibit glial, fate determination (Tomita et al., 2000; Morrison, 2001; Nieto et al., 2001).
The existence of a common ORN-sustentacular lineage has been suggested
by some, but not all, studies. For example, fate maps of single cells
in Xenopus olfactory placode demonstrated a common progenitor for ORNs and sustentacular cells, at least in early development (Burd et al., 1994). When adult rat OE was lesioned with
methyl bromide (which kills both neurons and sustentacular cells) and
allowed to regenerate, retroviral lineage mapping suggested the
existence of common ORN-sustentacular progenitors (Huard et al.,
1998). In contrast, in animals lesioned by olfactory bulbectomy (which
kills ORNs but not sustentacular cells), proliferation of cells that
express Mash1 and give rise to neurons increases greatly,
but proliferation of cells that become sustentacular cells does not
(Gordon et al., 1995; Calof et al., 1996a). Moreover, retroviral
lineage analysis of OE in unlesioned postnatal rats failed to find any
evidence for a common ORN-sustentacular lineage (Caggiano et al.,
1994).
Because sustentacular cells become a self-renewing population shortly
after their appearance in the OE, at ~E13.5 (Smart, 1971; Cuschieri
and Bannister, 1975; Weiler and Farbman, 1998), one explanation for
these data is that the common ORN-sustentacular progenitor generates
sustentacular cells only when "needed" to do so. Intriguingly, in
the OE there is strong evidence that neuronal production is repressed
by feedback signals from ORNs (Mumm et al., 1996; Wu et al., 2003).
Perhaps combinations of feedback signals from both ORNs and
sustentacular cells cooperatively tell bipotential progenitors not only
when to proliferate, but also what cell types to make.
| |
FOOTNOTES |
Received May 13, 2002; revised Dec. 11, 2002; accepted Dec. 12, 2002.
This work was supported by National Institutes of Health Grants DC03583
to A.L.C., NS26862 to A.D.L., HD38761 to A.D.L. and A.L.C., and the
March of Dimes Birth Defects Foundation (FY00-660 to A.L.C.). We thank
Carl Lagenaur for 12F8 anti-PSA-NCAM monoclonal antibody, Tom Curran
for reelin probe, and Shimako Kawauchi for help with
in situs.
Correspondence should be addressed to Anne L. Calof, Department of
Anatomy and Neurobiology, 364 Med Surge II, University of California,
Irvine, Irvine, CA 92697-1275. E-mail:
alcalof{at}uci.edu.
| |
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TOP
ABSTRACT
Introduction
