THE ANATOMICAL RECORD 295:769–775 (2012)

Development of the Juxta-Oral Organ in

Rat Embryo
J.R. MÉRIDA VELASCO,1* C. DE LA CUADRA BLANCO,1
2
AND J.A. MÉRIDA VELASCO
1
Departamento de Anatomı́a y Embriologı́a Humana II, Facultad de Medicina,
Universidad Complutense de Madrid, Spain
2
Departamento de Anatomı́a y Embriologı́a Humana, Facultad de Medicina, Universidad
de Granada, Spain

ABSTRACT
The aim of this work is to clarify the development and morphology of
the juxta-oral organ (JOO) in rat embryos from Day (E)14 to 19. Further-
more, in the region of the JOO, an analysis was made of the expression of
the monoclonal antibody HNK-1, which recognizes cranial neural-crest
cells. In this study, we report that JOO develops from an epithelial con-
densation at the end of the transverse groove of the primitive mouth at
E14. During E15, it invaginates and is disconnected from the oral epithe-
lium. At E16, the JOO forms an solid epithelial cord with three parts (an-
terior, middle, and posterior) and is related to the masseter, temporal,
medial pterygoid, and tensor veli palatini muscles. During E17-19, no sig-
nificant changes were detected in their position. Both the mesenchyme
caudal to the anlage of the JOO at E14, as well as the mesenchyme that
surrounds the bud of the JOO at E15, expressed positivity for HNK-1.
Our results suggest that the mesenchyme surrounding the JOO at E15
could emit some inductive signal for the JOO to reach its position at E16.
This work shows for the first time that the cranial neural-crest-derived
mesenchyme participates in the development of the JOO. Anat Rec,
295:769–775, 2012. V C 2012 Wiley Periodicals, Inc.

Key words: development; embryology; juxtaoral organ; HNK-1;

rat

INTRODUCTION to be condensed. However, we have observed that the mes-

Chievitz (1885), studying the development of the sali-
vary glands, described a thin epithelial structure near the
parotid duct, related to the buccal nerve. Ramsay (1935)
reported that this structure develops and disappears
before birth. Zenker’s group referred to the persistence of
a neuroepithelial organ that they termed the ‘‘juxtaoral
organ’’ (Salzer and Zenker, 1962). The presence of the
juxta-oral organ (JOO) has been reported in a wide range
of species such as fish, amphibians, reptiles, birds, and
mammals (Zenker and Halz, 1953; Grüneberg, 1971;
Jeanneret-Gris, 1980; Ito et al., 2009). In adult humans,
the JOO is a neuroepithelial structure located between
the temporal and buccinator muscles and near the pteri-
gomandibular raphe (Kramer and Zenker, 1974). We have
previously reported that the JOO in humans originated
from an epithelial condensation situated at the end of the
transverse groove of the primitive mouth, at Carnegie’s
stage 16. The surrounding mesenchyme does not appear
enchyme around the JOO was condensed by the 11th
week of development, to form its capsule (M
erida-Velasco
et al., 2005). Zenker’s group reported that the JOO is
organized in three layers or strata that surround the epi-
thelium: a thin inner ‘‘stratum fibrosum internum’’
formed by diverse layers of loose connective tissue; the
‘‘stratum fibrosum externum’’ made up of a dense connec-
tive tissue that surrounds the organ; and the nervous
fibres that form the ‘‘stratum nervosum.’’ We have
*Correspondence to: J.R. M
erida Velasco, Departamento de
Anatomı́a y Embriologı́a Humana II, Facultad de Medicina,
Universidad Complutense, Ciudad Universitaria s/n, 28040 Madrid,
Spain. Fax: þ34-91-394 71 87. E-mail: mvlopera@med.ucm.es
Received 5 May 2011; Accepted 21 November 2011.
DOI 10.1002/ar.22444
Published online 19 March 2012 in Wiley Online Library
(wileyonlinelibrary.com).

V
C 2012 WILEY PERIODICALS, INC.
770 VELASCO ET AL.
Fig. 1. Frontal sections stained with HE. Rat embryos at E14 (A, B),
E15 (C) and E16 (D, E, F). A, prospective region of the JOO. B, epithe-
lial condensation of the JOO at the end of the transverse groove of
the primitive month (arrows). C, in this stage the anlage of the JOO (J)
is not connected to the oral epithelium. D, the anterior portion of the
JOO (J) is related to the masseter muscle (MS). E, middle portion of
proposed a structural organization of the JOO in two
parts, the epithelium and the surrounding connective tis-
sue capsule, with the latter containing the vessels and the
nerves (M
erida-Velasco et al., 2005).
the JOO. F, posterior portion of the JOO. AT, auriculotemporal nerve;
B, buccal nerve; J, juxtaoral organ; M, mandible; MA, maxillary nerve;
MC, Meckel’s cartilage; MN, mandibular nerve; MS, masseter muscle;
G, trigeminal ganglion; OC, oral cavity; PM, medial pterygoid muscle;
T, temporalis muscle; TG, tooth germ; TV, tensor veli palatini muscle;
V, facial vein. A, C, D, E, F Bar ¼ 100lm; B Bar ¼ 20 lm.
The neural-crest cells are a transient migratory popula-
tion of stem cells derived from the dorsal neural folds at the
border between neural and non-neural ectoderm. The
migration of crest cells involves an epithelium-mesenchyme
 JUXTA-ORAL ORGAN IN RAT EMBRYO
Fig. 2. Schematic diagram of the arrangement of the JOO at E19 in
a lateral view. J, juxtaoral organ; T, temporalis muscle; MS, masseter
muscle; M, mandible.
transition, whereby mesenchyme cells become dispersed
along specific migratory pathways and located in their final
position. In the craniofacial region, they give rise to a wide
variety of cell types and tissues, including intramembra-
nous bone, cartilage, muscle, and nerves (Creuzet et al.,
2005; Noden and Trainor, 2005). The possible influence of
the cranial neural-crest-derived mesenchyme in the devel-
opment of the JOO has not been studied.
Despite decades of studies on JOO, very few descrip-
tions are available on the development and morphology
of this organ in rats. The aim of the present work was to
study the development and morphology of the JOO in
rat embryos at E14 to 19. In addition, we studied the
expression in the region of the JOO of the monoclonal
antibody HNK-1, which recognizes neural-crest cells
(Lipinski et al., 1983) and migration (Bronner-Fraser,
1986). Here, we report for the first time the participation
of the cranial neural-crest-derived mesenchyme in the
development of the JOO.
MATERIALS AND METHODS
Animals
The experimental protocol was approved by the Ethics
Committee for Animal Experimentation of University
Complutense of Madrid (Spain). Wistar rat embryos
from stages E14 to E19 (considering the fertilization day
as E0, based on vaginal cytology control) were used for
the study. The specimens were extracted from anaesthe-
tized and dissected mothers, and fixed in 4%
paraformaldehyde in 0.1 M phosphate buffered saline
(PBS) at pH 7.4 overnight at 4
C. Five specimens at
each stage were used. The embryos were dehydrated
and embedded in paraffin. Serial sections were cut at
6–8 lm thickness and mounted on poly-L-lysine pre-
coated slides. For orientation, sections were stained with
haematoxylin and eosin (HE).
Immunohistochemical Procedure
For the peroxidase antiperoxidase reaction, we used
the following procedures. The sections were pre-treated
with 0.3% hydrogen peroxide in PBS for 4 hr at room
temperature before washing in PBS (3  10 min). They
771
were incubated overnight at 4
C in 10% normal goat se-
rum (Sigma) and 0.2% Triton X-100 (Merck, Germany)
diluted in PBS. Sections were incubated in monoclonal
mouse antibody HNK-1 (Becton and Dickinson, CA) in a
1/20 dilution at 4
C overnight and then washed in PBS
(3  10 min). Afterward, they were again incubated at
4
C for 12 hr with horseradish peroxidase conjugated
secondary antibody (Chemicon) diluted 1/2500 and then
rinsed in PBS (3  10 min). The preparations were
treated with 3,30 Diaminobenzidine (Sigma). After final
rinsing in PBS (3  10 min), the preparations were
dehydrated in a series of alcohols, cleared in xylene and
mounted in Eukitt (O. Kindler GmbH and Co, Ger-
many). Controls were treated as above, but omitting the
incubation in the primary antibody solution, to demon-
strate that the secondary antibody reacted only with
this corresponding primary antibody.
RESULTS
Serial Sections Stained with HE
At E14 appeared a condensation of cuboidal epithelial
cells with rounded nuclei and scant cytoplasm. This con-
densation is visible at the end of the transverse groove
of the primitive mouth, near the mandibular nerve. This
epithelial condensation corresponds to the prospective
region of the JOO (Fig. 1A,B). At E15, the epithelial con-
densation invaginates and disconnects from the oral
epithelium, becoming surrounded by loose mesenchyme
and related to the buccal nerve (Fig. 1C). At E16, the
JOO forms a solid epithelial cord with no lumen. During
this stage, the anterior part of the JOO was related lat-
erally to the masseter muscle, surrounding its anterior
edge. In the middle part, it was related laterally to the
temporal muscle and dorsally to the blastemas of the
medial pterygoid and tensor veli palatini muscles (Fig.
1D–F). No significant changes were detected in the posi-
tion of the JOO during E17-19. The thickest portion was
the anterior, which thinned in the posterior direction.
The arrangement of the JOO at E19 is represented in
Figure 2.
Immunohistochemistry
At E14, HNK-1 positivity was noted in some zones of
the trigeminal ganglion and in the mesenchyme that
surrounded both the mandibular nerve as well as the
branches of the maxillary artery (Fig. 3A–D). The man-
dibular nerve and the mesenchyme located between the
nerve and the end of the transverse groove of the primi-
tive mouth showed positivity for HNK-1 (Fig. 3E–G).
At E15, the loose mesenchyme surrounding the JOO
and the buccal nerve proved HNK-1 positive, whereas
the JOO was negative (Fig. 4A–F). At E18, the buccal
nerve was HNK-1 positive and a branch of it innerved
the JOO (Fig. 5A–D). At all the stages analyzed, the
JOO proved HNK-1 negative.
DISCUSSION
The results of this study show that the development
of the JOO in the rat begins at E14, with the epithelial
condensation of the end of the transverse groove of the
primitive mouth, relating it to the mandibular nerve.
This epithelial condensation corresponds to the
772 VELASCO ET AL.
Fig. 3. Rat embryo E14. Frontal sections of the region of the tri-
geminal ganglion (A, B, C, D). Frontal section of the region of the end
of the transverse groove of the primitive month (E, F, G). HE staining
are shown in A and E; HNK-1 immunoperoxidaxe labelling are shown
from B, C, D, F and G. B, the trigeminal ganglion (G) is HNK-1 posi-
prospective region of the JOO; the surrounding mesen-
chyme was not condensed. At E15, the JOO was
disconnected from the epithelium of origin. In mice, the
JOO appeared in the same region at E12 (Ito et al.,
2009). Previously, we have observed an epithelial thick-
ening in the proximity of the mandibular nerve, at
tive. C, D, F and G, the mandibular nerve (MN) and the mesenchyme
cells are HNK-1 positive (arrows). A, maxillary artery branch; OC, oral
cavity. TO, tongue. A, B, C Bar ¼ 100 lm. D, E, F Bar ¼ 50 lm. G
Bar ¼ 20 lm.
Carnegie’s stage 16 of human embryos (M
erida-Velasco
et al., 2005). Grüneberg (1971) studying mice, found
three parts in the JOO, the cauda, corpus, and cervix,
whereas Ito et al. (2009) called them anterior, middle,
and posterior portions, respectively. Examining rats, we
observed that the location of JOO is similar, as the
 JUXTA-ORAL ORGAN IN RAT EMBRYO
Fig. 4. Rat embryo E15. Frontal sections of the region JOO. HE
staining sections are shown in A, C, and E. HNK-1 immunoperoxidaxe
labelling are shown in B, D, and F. Nasal capsule (NC), nasal septum
(S), facial nerve (F) and hypoglossal nerve (H) are HNK-1 positive in B.
anterior part is related to the masseter muscle; the mid-
dle part to the temporal muscle, whereas the posterior
part is related to the medial pterygoid and the tensor
veli palatini muscles, being located near the posterior
part of the tongue. In addition, we verified that the JOO
is ticker in the anterior portion than the posterior.
773
B, buccal nerve; J, juxtaoral organ; M, mandible; MC, Meckel’s carti-
lage; NF, nasal fossa; OC, oral cavity; TG, tooth germ; TO, tongue;
VN, vomeronasal organ. Mesenchyme cells HNK-1 positive (arrows) in
D and F. A, B Bar ¼ 200 lm. C, D Bar ¼ 100 lm. E, F Bar ¼ 50 lm.
In this study, at E14, the trigeminal ganglion showed
zones of positivity for HNK-1. Also, the mesenchyme
surrounding both the mandibular nerve as well as the
branches of the maxillary artery was positive. Probably,
the positive zones came from the cranial neural-crest
(Noden, 1993). It has been reported that in rats the
774 VELASCO ET AL.
Fig. 5. Rat embryo E18. Frontal sections of the region JOO. HE
staining sections are shown in A and C. HNK-1 immunoperoxidaxe
labelling are shown in B and D. B, buccal nerve; E, eye; J, juxtaoral
organ; NP, nasopharynx; OC, oral cavity; ON, optic nerve; R, retina
cranial ganglia and nerves were HNK-1 positive (Louryan
et al., 1996; Nagase et al., 2003). Thus, in human
(O’Rahilly and Müller, 2007) and chicken embryos (Shige-
tani et al., 2008), it has been reported that the trigeminal
ganglion originated from the neural crest and from the
trigeminal placode (Knabe et al., 2009). Probably, the
parts of the trigeminal ganglion that originate from the
trigeminal placode correspond to the parts that were
HNK-1 negative.
Although the molecular mechanisms of the develop-
ment of the JOO are not known, these appear to differ
from the classical epithelium-mesenchyme interaction of
other epithelial organs (M
erida-Velasco et al, 2005; Ito
et al., 2009). We have reported that in human develop-
ment the mesenchyme surrounding the JOO is loose,
while that surrounding the bud of the parotid gland is
condensed. However, during the 11th-12th week of devel-
opment, the mesenchyme surrounding the JOO appeared
condensed, constituting a anlage of the capsule and dur-
ing the 13th week the parenchyma of the JOO showed
buds (M
erida-Velasco et al., 2005). This may suggest that
the formation of the capsule could be a criterion of JOO
maturity, although in the present study the mesenchyme
(layer of nerve fibres) TO, tongue; Buccal nerve branch (arrows in D).
Buccal nerve branch (arrow head in D). The squared area in B is mag-
nified in D. A, B Bar ¼ 200 lm. C, D Bar ¼ 20 lm.
surrounding the JOO was not condensed in the stages an-
alyzed and there was no identifiable capsule.
The mesenchyme caudal to the prospective region of
the JOO at E14, as well as the mesenchyme that sur-
rounded both the JOO and the buccal nerve at E15,
were all HNK-1 positive. This suggests that the mesen-
chyme was derived from the cranial neural-crest and
migrated accompanying the mandibular nerve, to be
located near the epithelial condensation that would give
rise to the JOO. Recent studies suggest that the fate of
the neural crest is strongly influenced by environmental
cues. Tooth development is a clear example of the con-
sistent shift of instructive signal between orofacial
epithelium and the cranial neural-crest-derived mesen-
chyme (Chai et al., 2000; Chai and Maxson, 2006).
Tissue-recombination experiments show that the induc-
tive tooth signal first occurs in the oral epithelium and
then shifts into the underlying cranial neural-crest-
derived mesenchyme at a later development stage (Mina
and Kollar, 1987). In our study, the epithelial condensa-
tion of the primitive mouth at the prospective region of
the JOO appeared at E14, and positive HNK-1 cells sub-
sequently migrated toward this region. This suggests
 JUXTA-ORAL ORGAN IN RAT EMBRYO
that the prospective area of the JOO could establish
some type of signal for the migration of the cranial neu-
ral-crest-derived mesenchyme.
In addition, during E15, the mesenchyme surrounding
the JOO was HNK-1 positive, while it was negative at
E16–19. However, during these stages, some structures
such as the buccal nerve, maxillary nerve, optic nerve,
retina (layer of nerve fibres), and cranial dura mater
proved positive, this being evidence that the antibody
functioned. Our results suggest that the mesenchyme
surrounding the JOO during E15, which gives rise to
the neural crest, with migratory properties, could emit
some sort of inductive signal for the JOO to reach its
position during E16.
The function of the JOO is unknown. In humans, the
possibility of a receptor function of the JOO has been
raised (D’Andrea et al., 1999; Pantanowitz and Balogh,
2003; M
erida-Velasco et al., 2005). Recently, Ito et al.
(2009), studying mice, considered the JOO to be a sen-
sory receptor. Developmentally, mechanoreceptive
neuromasts and electroreceptive ampullary organs have
been suggested to have arisen from epithelial placodes
in amphibians and fish (Northcutt et al., 1995; Gibbs
and Northcutt, 2004). However, cell-lineage studies have
shown that mechanoreceptor development in amphibians
and teleosts also involves neural-crest cells (Collazo
et al., 1994). Recently, Freitas et al. (2006) reported that
the electroreceptors of sharks may have a dual origin:
from an epithelial condensation and from the cranial
neural-crest-derived mesenchyme. The similar develop-
ment of JOO in rats could suggest that this plays a role
as sensory receptor.
ACKNOWLEDGEMENT
We wish to express our gratitude to the following for
their technical assistance in the preparation of this pa-
per: Mrs Purificaci
on Chimeno; Mrs Araceli Garcı́a; Mrs
Montserrat Juanilla and Mr Jes
us de la Calle.
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