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Expression of vomeronasal receptor genes in Xenopus laevis.
Hagino-Yamagishi K
,
Moriya K
,
Kubo H
,
Wakabayashi Y
,
Isobe N
,
Saito S
,
Ichikawa M
,
Yazaki K
.
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In the course of evolution, the vomeronasal organ (VNO) first appeared in amphibians. To understand the relationship between the VNO and the vomeronasal receptors, we isolated and analyzed the expression of the vomeronasal receptor genes of Xenopus laevis. We identified genes of the Xenopus V2R receptor family, which are predominantly expressed throughout the sensory epithelium of the VNO. The G-protein Go, which is coexpressed with V2Rs in the rodent VNO, was also extensively expressed throughout the vomeronasal sensory epithelium. These results strongly suggest that the V2Rs and Go are coexpressed in the vomeronasal receptor cells. The predominant expression of the Xenopus V2R families and the coexpression of the V2Rs and Go imply that V2Rs play important roles in the sensory transduction of Xenopus VNO. We found that these receptors were expressed not only in the VNO, but also in the posterolateral epithelial area of the principal cavity (PLPC). Electron microscopic study revealed that the epithelium of the PLPC is more like that of the VNO than that of the principal and the middle cavity. These results suggest that in adult Xenopus the V2Rs analyzed so far are predominantly expressed in the vomeronasal and vomeronasal-like epithelium. The analysis of V2R expression in Xenopus larvae demonstrates that V2Rs are predominantly expressed in the VNO even before metamorphosis.
Fig. 1. Deduced amino acid sequence of the newly isolated Xenopus
vomeronasal receptor cDNA. Aligned deduced amino acid sequences
of goldfish 5-24 (Speca et al., 1999), mouse V2R1 (Matsunami
and Buck, 1997), and newly isolated Xenopus xV2R1 vomeronasal
receptors. Predicted positions of the seven transmembrane domains
are underlined. The identical residues are shown in white letters on a
black background. Extracellular cysteine residues that are also
present in mGluRs and mouse V2Rs are marked (*).
Fig. 2. Expression of Xenopus V2Rs in various tissues. Expression
of Xenopus vomeronasal receptors (V2Rs) was analyzed by RT-PCR as
described in Materials and Methods. RT-PCR products derived from
total RNA isolated from the olfactory epithelium and vomeronasal
organ (lanes 3, 4), olfactory bulb (lane 5), brain (lane 6), heart (lane 7),
intestine (lane 8), stomach (lane 9), and kidney (lane 10) were compared
to the PCR product derived from Xenopus genomic DNA (positive
control) (lane 2). As a negative control, the cDNA synthesis was
performed without reverse transcriptase (lane 4). The 440-bp and
160-bp PCR products (indicated by arrows) correspond to Xenopus
V2Rs and -actin cDNA, respectively. The marker used was the
HaeIII-digested fX174 DNA (lane 1).
Fig. 3. Analysis of Xenopus V2R expression in the vomeronasal
organs by in situ hybridization. Cross sections of Xenopus vomeronasal
epithelium were stained with hematoxylin (A). The cross
sections were hybridized to digoxigenin-labeled antisense
probes which were derived from clones A-1 (B), B-1 (C), C-1 (D),
E-1 (E), xV2R1 (F), a mixture of A-1, C-1, and E-1 (G), and sense
probes derived from a mixture of A-1, C-1, and E-1 (H). RE, receptor
cell layer; SU, supporting cell layer; VL, lumen of the VNO. Black
spots around the outside of the VNO in A–H (for example, arrow
in F) are melanocyte aggregates. Scale bars 50 m.
Fig. 4. Expression of G-proteins in the vomeronasal organ (VNO),
and expression of the Xenopus V2R in the principal cavity (PC) and
the middle cavity (MC) of the nasal capsule. VNO cross-sections were
hybridized to Go (A) and Gi2 (B) antisense probes, and to Go (C) and
Gi2 (D) sense probes (negative controls). Some cells in the MC were
hybridized to the Gi2-antisense probe (B’) (positive control for the
Gi2-antisense probe). The antisense Xenopus V2R probes derived
from clones A-1, C-1, and E-1 were mixed, and sections of Xenopus PC
(E) and MC (F) epithelia were hybridized to these probes. An arrow
indicates the V2R-expressing cell (F). Scale bars 50 m.
Fig. 5. Expression of Xenopus
V2Rs in the posterolateral epithelial
area of the principal cavity.
Photographs of hematoxylinstained
transverse sections of Xenopus
laevis upper jaw at intervals
of 400 m (A). Arrow indicates the
V2R-expressing region (A-3). PC,
Principal cavity; MC, middle cavity;
VNO, vomeronasal organ.
Cross-sections of Xenopus nose
were hybridized to digoxigeninlabeled
antisense probes prepared
from clones E-1 (B), xV2R1 (C),
and Go (D). Arrows indicate the
xV2R1-expressing cells (C). Scale
bars 1 mm in A; 100 m in B–D.
Fig. 6. Electron micrographs of
the posterolateral epithelial area
of the principal cavity. A: Microvillar
receptor cells (re) and ciliated
supporting cells (sp) were observed.
Microvilli (arrows) on a microvillar
receptor cells are indicated.
Scale bar 1 m. B: Higher
magnification of luminal surface of
microvillar receptor cell. Microvilli
are indicated (arrows). Dense border
present in cell-to-cell junction
(arrowhead) indicates the junctional
complex. Scale bar 500
nm. C: Higher magnification of luminal
surface of ciliated supporting
cells. Microvillus (large arrow),
kinocilia (arrowheads), ciliary
rootlets (double arrow), and ciliary
basal bodies (small arrows) are indicated.
Scale bars 500 nm.
Fig. 7. Expression of Xenopus V2Rs in the olfactory and vomeronasal
epithelial cells of Xenopus larvae. Cross sections of Xenopus
larvae at stage 45 (A) and stage 50 (B–D) were hybridized to
digoxigenin-labeled antisense probes prepared from clones E-1
(A,B,D) and xV2R1 (C). Arrows indicate the receptor-expressing cells.
OE, olfactory epithelium; VNO, vomeronasal organ. Scale bars 50
um.
v2ra-1 (vomeronasal receptor A-1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 66, sagittal sections of the olfactory region.
v2rb-1 (vomeronasal receptor B-1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 66, sagittal sections of the olfactory region.
v2rc-1 (vomeronasal receptor C-1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 66, sagittal sections of the olfactory region.
xv2r1 (pheromone receptor-like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 66, sagittal sections of the olfactory region.
