First published online March 1, 2004
doi: 10.1242/10.1242/dev.01009
Development 131, 1377-1388 (2004)
Published by The Company of Biologists 2004
Genetic disruptions of O/E2 and O/E3 genes reveal involvement in olfactory receptor neuron projection
Song S. Wang1,2,3,
Joseph W. Lewcock1,2,
Paul Feinstein4,
Peter Mombaerts4 and
Randall R. Reed1,2,3,*
1 The Howard Hughes Medical Institute, The Johns Hopkins University School of
Medicine, 725 North Wolfe Street, PCTB 818, Baltimore, MD 21205, USA
2 Department of Molecular Biology and Genetics, The Johns Hopkins University
School of Medicine, 725 North Wolfe Street, PCTB 818, Baltimore, MD 21205,
USA
3 Department of Neuroscience, The Johns Hopkins University School of Medicine,
725 North Wolfe Street, PCTB 818, Baltimore, MD 21205, USA
4 The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
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Fig. 1. Targeted disruption of O/E genes. (A) Schematic of O/E2
targeting strategy. The taulacZ-pA-HSV-tk(x)-pgk-neo cassette was
inserted into the 5' UTR of the O/E2 gene, replacing the first
six exons encoding the first 185 amino acids of the O/E2 protein, and placing
the tau-lacZ-pA cassette under the transcriptional control of the
endogenous O/E2 promoter. The thymidine kinase (tk) cassette
was inactivated by deleting a 476 bp BstBI-NruI fragment to
permit transmission through the male germline. Mice were generated with ES
cells carrying mutation in the O/E2 locus and mated to cre-expressing
mice to remove the HSV-tk( )-pgk-neo cassette flanked
by loxP sites. The position of EcoRI restriction enzyme recognition
sites and the location of the probe used for Southern blot confirmation of
homologous recombination are indicated. (B) Schematic of O/E3
targeting strategy. The strategy is similar to the O/E2 targeting
strategy with the following changes. A tau-GFP-pA reporter gene was
used in the place of tau-lacZ-pA cassette, and five exons of the
O/E3 gene encoding the first 162 amino acids of the O/E3 protein were
replaced. (C) Southern blot analysis of the O/E2 alleles. Genomic DNA
of O/E2 mutant littermates was digested with EcoRI
restriction enzyme, separated by agarose-gel filtration and subjected to
Southern blot analysis. A wild-type O/E2 allele yields a 8 kb
hybridization signal and a mutated O/E2 allele gives a 6 kb signal.
(D) Southern blot analysis of the O/E3 alleles. A wild-type
O/E3 allele gives a 7.5 kb hybridization signal and a mutated
O/E3 allele gives a 3 kb hybridization signal. (E) In situ
hybridization of olfactory epithelium sections of neonatal O/E2
heterozygous and homozygous animals with O/E1, O/E2 and O/E3 probes. (F) In
situ hybridization of olfactory epithelium sections of neonatal O/E3
heterozygous and homozygous animals with O/E1, O/E2 and O/E3 probes.
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Fig. 2. Expression of Tau-reporters mimic endogenous O/E pattern. (A,C)
Mid-sagittal view of an adult O/E2 heterozygous animal after X-gal
staining. ß-galactosidase activity was present in the olfactory
epithelium and bulb (A), vomeronasal organ (C) and axon fibers projecting from
these two structures. (B,D) Mid-sagittal view of an adult O/E3
heterozygous animal. GFP fluorescence was visible in the olfactory epithelium
and bulb (B), vomeronasal organ (D), and axon fibers projecting from these two
structures. (E) X-gal staining of olfactory epithelium section of an
O/E2lacZ/lacZ animal showing ß-gal activity in the
ORN cell bodies and axon bundles. (F) X-gal staining of OB section of
O/E2lacZ/+ animal showing ß-gal activity in ORN axons
projecting to the olfactory glomeruli. (G) An olfactory epithelium section of
an O/E3GFP/+ animal showing GFP fluorescence in the ORN
cell bodies and axon bundles. (H) An OB section of
O/E3GFP/+ animal showing GFP fluorescence in ORN axons
projecting to the olfactory glomeruli. (I,J) Whole-mount X-gal staining of an
E11 O/E2lacZ/+ mouse embryo showing ß-gal expression
in the neural tissues. (K) Two-color confocal image of OE from heterozygous
OE3 mouse. Intrinsic GFP localization is shown in green and neuronal specific
tubulin shown in red. Two cells near the basal lamina that express only the
GFP reporter are indicated by arrows.
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Fig. 3. Neonatal O/E2lacZ/lacZ mice exhibit ORN projection
defect. (A) Whole-mount X-gal staining of O/E2lacZ/+ and
O/E2lacZ/lacZ neonates showing the ORN projection patterns
to the OB. At P1 in O/E2lacZ/+ animals, the ß-gal
positive ORN axons cover most of the OB surface, but the
O/E2lacZ/lacZ mice lack projections to the lateral and
dorsal regions of their OB. In addition to the projection phenotype, the OB of
neonatal O/E2lacZ/lacZ animals are significantly smaller
and rounder in shape than those of the heterozygous littermates. Similar
results were observed in additional O/E2lacZ/lacZ mice
(n=8). (B) ß-Galactosidase staining and OMP immunohistochemistry
of coronal sections through the OB of O/E2lacZ/+ and
O/E2lacZ/lacZ neonates showing the ORN projection patterns
to the OB. The sections are matched by the morphology of the olfactory
turbinates. In the sections of O/E2 heterozygous neonate, the
ß-gal and OMP-positive ORN axons covers most of the OB surface, but the
O/E2lacZ/lacZ animals display no apparent projections to
the lateral and dorsal regions of their OB. In addition, the sections also
demonstrate the shortening of the OB in the O/E2lacZ/lacZ
animals. In all panels, dorsal is at the top and medial is towards the
right.
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Fig. 4. Adult O/E3 mutants exhibit ORN projection defect. (A) The ORN
projection to the OB was visualized by O/E3-directed GFP fluorescence
in heterozygous and O/E3GFP/GFP mice. The absence of ORN
axons on the dorsal surface of O/E3GFP/GFP OB can be seen
in the whole-mount view and in coronal sections. Similar results were obtained
in five independent mice of each genotype. Similar to the observation in
O/E2 mutant animals, the OBs of adult O/E3GFP/GFP
mice are slightly smaller and rounder in shape than that of their heterozygous
littermates. (B) OMP immunohistochemistry of coronal sections through the OB
of O/E3 heterozygous and homozygous neonates showing the ORN
projection patterns to the OB. Sections from both anterior and posterior OB
are shown. In both animals, the ORN axons cover the entire OB. However,
stronger anti-OMP immunoreactivity was seen on the dorsal and lateral OB of
the O/E3GFP/GFP neonate, and thinning of the external
plexiform layer on the dorsal aspect of O/E3GFP/GFP OB was
observed, indicating a possible prelude to the adult phenotype. In all panels,
dorsal is towards the top and medial is towards the right.
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Fig. 5. Functional connections are established in the OB of adult
O/E3GFP/GFP mutants. (A) The ORN projection to dorsal OB
and olfactory glomeruli formation in this region were visualized by GFP
fluorescence and OMP immunofluorescence. The patterns of GFP and OMP in the
coronal sections clearly show the absence of olfactory glomeruli in the dorsal
aspect of O/E3GFP/GFP mutant OB. (B) Tyrosine hydroxylase
(Th) expression in the periglomerular neurons is dependent on innervation and
activity from the olfactory epithelium. Immunofluorescence demonstrates that
Th expression overlaps the GFP-positive glomeruli and suggests that ORNs
transduce signals and form functional connections within the OB of
O/E3GFP/GFP animals. (C) Characterization of OB neurons in
O/E3GFP/GFP mice. The soma of mitral, tufted and
periglomerular cells are visualized with a fluorescent Nissl stain in
heterozygous and homozygous O/E3GFP/GFP mice. Although
there is considerable disorganization of the glomerular layer, many neurons
are present. The intensity of dendritic marker Map2 staining is considerably
diminished in O/E3GFP/GFP animals, consistent
with the thinning of the external plexiform layer on the dorsal surface. The
intrinsic GFP labeling of ORN axons and their convergence into glomeruli is
shown in green.
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Fig. 6. O/E2lacZ/+/O/E3GFP/+ double heterozygous
animals exhibit similar projection defects independent of Tau overexpression.
(A) The ORN projection patterns on dorsal OB of adult
O/E2lacZ/+ and O/E3GFP/+ mice were
visualized by whole-mount X-gal staining. ORN axons and glomeruli are visible
on the entire dorsal surface of the OB. (B) X-gal staining pattern of an
O/E2lacZ/+/O/E3GFP/+ animal show areas where
ORN axons and glomeruli are absent. (C) X-gal staining pattern of an
O/E3GFP/+ animal carrying two OMP-tau-lacZ
alleles reveals a normal projection pattern, indicating that the extra gene
dose of tau-ß-galactosidase from the OMP-taulacZ alleles does
not cause a projection defect. (D) Coronal sections of the OB from an animal
with the same genotype as the one described in C show normal projections to
the dorsal surface. (E) Coronal sections of the OB from an
O/E3GFP/GFP animal carrying one OMP-tau-lacZ
allele show the same projection defect seen in O/E3GFP/GFP
homozygous animals.
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Fig. 7. Positions of P2 glomeruli are shifted ventrally. (A) Projections of ORNs
expressing the P2 odorant receptor were visualized by X-gal staining in an
O/E3GFP/+ mouse carrying a P2lacZ
allele. (B) In agreement with the previously described pattern, axons from
P2-expressing ORNs project dorsally before converging to a defined location on
the medial surface of the OB in O/E3GFP/+ animals. In an
O/E3GFP/GFP mouse carrying a P2lacZ
allele, very few axons were visible on the medial surface of the OB indicating
a change in projection pattern. (C,D) X-gal staining on coronal sections of OB
showed convergence of P2-expressing axons to defined glomeruli on the medial
aspect of OB. X-gal signal was pseudocolored in white for clear visualization.
The sections were also stained with DAPI (blue) to mark the layers of the OB,
and ORN axons were visualized by GFP fluorescence. P2-expressing axons in
O/E3GFP/GFP homozygous animals converge to a more ventral
position relative to their heterozygous littermates. (E,F) Coronal sections of
the OB were treated as described in C to show convergence of P2-expressing
axons to defined glomeruli on the lateral aspect of OB. Similarly,
P2-expressing axons in O/E3 homozygous animals converge to a more
ventral position relative to their heterozygous littermates.
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Fig. 8. The dorsal M72 glomeruli are retained in O/E3GFP/GFP
homozygous mice. The tau-lacZ reporter-tagged receptor M72 converges to a
single lateral glomerulus in O/E3GFP/+ and
O/E3GFP/GFP homozygous animals. The bulb was mounted, GFP
was imaged by confocal microscopy and the bulb stained to visualize
lacZ-containing axons. An overlay of the two images, registered with
landmarks in the bulb, reveals that the M72 glomerulus is present in
O/E3GFP/GFP homozygous mice.
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© The Company of Biologists Ltd 2004
