Translational Research in Anatomy 19 (2020) 100063

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Translational Research in Anatomy

journal homepage: www.elsevier.com/locate/tria

Primary cilia in the Syrian hamster biliary tract: Bile flow antennae and T
outlooks about signaling on the hepato-biliary-pancreatic stem cells
J. Gilloteauxa,b,c,∗
a
Department of Anatomical Sciences, St George's University International School of Medicine, KB Taylor Global Scholar's Program at UNN, School of Health and Life
Sciences, Newcastle Upon Tyne, NE1 8ST, United Kingdom
b
Department of Surgery, Summa Research Foundation, Akron, OH, USA
c
Unité de Recherches en Physiologie Moléculaire (URPHyM-Narilis), Départment de Médecine, Université of Namur, Namur, Belgium

ARTICLE INFO ABSTRACT

Keywords: Background: Microscopic anatomy investigations about the liver and biliary tract of the Syrian hamster, in re-
Liver ference with human structure, functions and defects in biliary tract and other organs.
Bile Methods: Electron microscopy technique and literature review.
Cholangiocyte Results: The author reports the finding of primary cilia in the biliary outflow channels. By places, the widest
Primary cilium
ducts also show blunted, or even enclosed, primary cilia, either as remnants or growing primary cilia.
Syrian hamster
Conclusions: The report should trigger further translational interest in human hepatology because, as seen in
rodents, these sensing cell appendages, transducing antennae of biliary flow and quality, could feed back to be
part of regulation of the hepatic tract motility and the secretory liver. There, adjacent oval or intermediate cells
could be, through some intercellular transduction mechanism(s), influenced to differentiate into either cho-
langiocytes or hepatocytes.

1. Introduction cilium among the apical microvillar cell specializations. Zimmerman, in

Morphological and physiologic changes of the gall bladder and liver
induced by sex steroid hormone treatment of the female and male
Syrian hamster [1–9] appeared to match those involved in lipid and bile
metabolism of the human, where cholelithiasis and other defects would
occur, including mechanic changes of the cystic or common bile duct
lumen and spiral, likely due to passage of calculi, as abraded epithelial
surfaces compared to those found in the normal organ [11]. Ad-
ditionally, if females exhibit a higher prevalence of lithogenic bile and
gallbladder disease compared with males, this can suggest that sex
hormones may be involved in the pathogenesis of chronic and acute
cholecystitis, especially the progesterone and medroxyprogesterone
principally used as contraceptive and treatment for endometriosis;
these compounds remove or deplete the expression (mRNA) of the
cholesterol hydroxylase from the liver metabolism [13]. Another recent
publication [14] seemed to not have considered our series of data
dealing with a similar topic about the sex hormone influences [1–13].
These aforementioned fine structure and functional investigations
[1–13] allowed us to detect cholangiocytes lining the bile channels of
the untreated hamster liver that demonstrated some single, apical
1898, was first to describe these peculiar appendages in epithelia with
light microscopy [15] while Sorokin has been credited to have first
found them on fibroblasts and smooth muscle cells [16]. Meanwhile,
the same structures have been given diverse names: ‘cilia’ in rodents
[17], ‘cilium’ in the bile ductule (Hering canal) of human fetus [18] and
adult liver [19] ‘cholangiocilia’ [20], ‘oligocilia’ [21] or ‘single cilia’
[22–24]. Found again in the bile ducts of monkeys [25] and birds [26],
similar ‘cilia’ have been recognized in the collecting ducts of the ne-
phron [27]. In textbooks, the primary cilium was first coined as ‘solitary
cilium’ as well as in the bile ducts, rete testis, intercalated ducts of
pancreatic glands [28]. These peculiar cell's antennae were also com-
piled from numerous publications at the Wadsworth Center in Albany,
New York by Sam S. Bowser (as referred in www.bowserlab_org/
primary cilia/cilialist.html). The list was revised and maintained by
Wheatley [29–31] and others at the site www.primary-cilium.co.uk.
The former e-link already contains a huge list of references about pri-
mary cilia found in invertebrate and vertebrate cell types, tissues and
organs, normal and pathologic ones and another link was more recently
developed [30]. In addition, like for oldest references that included all
sorts of epithelial-derived cells [29–47], one also noticed oval cells,

∗
Department of Anatomical Sciences, St George's University International School of Medicine, KB Taylor Global Scholar's Program at UNN, School of Health and
Life Sciences, Newcastle Upon Tyne, NE1 8ST, United Kingdom.
E-mail addresses: jacques.gilloteaux@unamur.be, jgilloteaux@sgu.edu.

https://doi.org/10.1016/j.tria.2020.100063
Received 20 November 2019; Received in revised form 9 January 2020; Accepted 9 January 2020
Available online 17 January 2020
2214-854X/ © 2020 Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
J. Gilloteaux Translational Research in Anatomy 19 (2020) 100063

adjacent to the interlobular spaces [37], the so-called ‘portal triad’,

better named ‘portal space of Kiernan’, were observed among the biliary
tracts lined by cholangiocytes [49–54,57,58]. It is also because the
persistent interest formed by those reviews of the topic [38–48] that
gave us grounds to review our collection of micrographs and generate
this report. We believe that our observations can fulfill some of the
previous suggestions [30–32,45,59,60], to add more comparative data
to bring up about the biological significance of these peculiar cell,
sensing appendages.
This report complements and comforts some early data on primary
cilia and some fine structure aspects of the cholangiocytes in this small
mammal [5], confirming the short survey of Gilroy and collaborators
[61] that included the hypothesis of a sensory function as ‘antennae’ of
those ‘solitary cilia’ found in the bile ductules of the pig. Our mor-
phological data also supplement information about the fine structure of
the smallest interlobular bile channels only illustrated in human de-
velopment [17,62]. Based on the aforementioned studies and on this
report, one can further confirm that the primary cilia of the cho-
langiocytes of the bile duct system, located in a way similar to those
studied in the pancreatic ducts, could likely be of important mechano-,
osmo- and chemosensory function through transduction activities reg-
ulating cholangiocyte aquaporin channels and bicarbonate secretion
along the bile outflow system of the exocrine liver. Those functions
could also relate to homeostatic maintenance of liver cell generation Fig. 1. A-D: Liver tissue from 60 days old male Syrian hamster. A: Hepatocytes
through local, junctional complex or circulating signaling ligands with from distal acinar zone III displaying several bile canaliculi (arrowheads). A
oval or intermediate cells. small sinusoid can be noticed in the upper left with an adjacent, perisinusoidal,
storage or stellate of Küpffer (s) with two large fatty inclusions. Scale in A 5 μm.
2. Materials and methods B: Hepatocytes from acinar zone II show a cord organization and two bile ca-
naliculi (arrowheads). Scale is 5 μm. C: Example of hepatocyte from acinar zone
Specimens obtained from five 60 day old male Syrian hamsters (Syr II displaying a typical, spheroid and euchromatic nucleus in a cytoplasm rich in
lak random bred, Charles River Co, Neder-over-Heembeek,Belgium), mitochondria, smooth ER and RER with empty glycogen patches (g) caused by a
kept caged with rodent chow and water ad libitum under 20-21 °C fixation without alcohol. Scale is 1 μm. Insert contains a typical peroxisome (p)
adjacent to a nuclear envelope. Scale 200 nm. D: Mitochondria with inter-
temperature, under light and dark cycles of 12 h/12 h in the Animal
spersed and aggregate of peroxisomes with an electron dense, urate oxidase
Care Unit of the Anderlecht Campus Research facility of the Medical
paracrystal showing as single or crossed, curve shapes and almost bisecting the
School Research facility, Université Libre de Bruxelles (ULB), Brussels organelles with electron dense, residual bodies in the vicinity of a bile canaliculi
(Belgium). Males were used due to suggested and verified influences of (bc). Scale is 1 μm.
estrogen on biliary structures [34] and composition [63,64] and
through data collected from our other investigations [1–10]. Hamsters
acinus and quite similar to those found in zones II). Those hepatocytes
were cared and also utilized also for multiple studies involving cardi-
appear with diverse in electron density, as uninucleate or binucleate
ovascular and hepatologic studies in accordance to laboratory care and
cells. The most centrolubularly-located hepatocytes contain typical or-
approval by both the NEOUCOM Comparative Animal Care Committee,
ganelles interspersed in a cytoplasm rich in glycogen accumulated as
Rootstown OH following the National Institutes of Health directives
circular-shaped fields of cytoplasm without showing classic alpha ro-
(NIH Publications No. 8023, revised 1978) and the EU Directive for
settes deposits inside encircling mitochondria. Those mitochondria can
animal experiments in the Animal Research Committee of the ULB
be found associated with dispersed peroxisomes, pigments and a few
Research Committee facility. Animals were anesthetized by an in-
lipid droplets. The ovoid-to-round mitochondria show either a con-
traperitoneal injection of sodium barbital (5–7 mg/100 gm b. w.) and
tracted, condensed matrix or even a pale, dilated matrix even though
after cervical dislocation, liver and gallbladder were excised, sliced,
tissues were all fixed simultaneously. These mitochondria are char-
washed in Tyrode saline to remove blood, then fixed in 3.5% buffered
acteristically almost completely surrounded by one or two scythe-
glutaraldehyde solution containing 0.1 M Na cacodylate, pH 7.35, at
shaped cisterns of the rough endoplasmic reticulum (RER). The nuclei
room temperature for 15 min, and then the same fixation was continued
are typically spheroid-shaped, euchromatic and can be eccentric
for 2 h at 4 °C. Washed in buffered sucrose solution, specimens were
(Fig. 1A). Not far away from the central venule, the spaces of Disse can
then postfixed in 1.5% aqueous osmium tetroxide solution and pro-
be noted containing perisinusoidal stellate cells (historically, first dis-
cessed for transmission (TEM) electron microscopy after embedment in
covered by Küpffer along the others, namely the macrophages of the
PolyBed epoxy resin (Polysciences, Warrington, PA.). Ultrathin sections
liver) better known as fat-storing or Ito cells) because showing small to
were collected on 75- and 100-mesh hexagonal copper grids (SPI, West
large fatty inclusions (Fig. 1 A). The hepatocytes are also forming bile
Chester, PA.) and then contrasted by uranyl acetate and lead citrate
canaliculi of 1–2 μm in diameter (arrows in Fig. 1 A). In Fig. 1 B he-
prior to examination in a Jeol 100 S electron microscope.
patocytes of the outermost, acinar zone III or zone II appear to initiate a
cord-like arrangement. There the cytoplasm shows a few lipid inclu-
3. Results
sions, with all the other aforementioned organelles. Fig. 1C depicts a
typical hepatocyte of zone II rich in RER and mitochondria with some
3.1. The liver tissues and bile canaliculi
autophagosomes and peroxisomes. The insert in the same Fig. 1C il-
lustrates a typical aspect of Syrian hamster peroxisomes, sometime not
Following ultrastructural observations, Fig. 1 A –D exemplifies with
only located adjacent to mitochondria but also adjacent to the nuclear
low magnification typical histological aspects of adjacent hepatocytes
envelope. Peroxisomes can be easily recognized because they appear
of the Syrian hamster as observed close to the central areas of a lobule,
ovoid, from 0.3 to 1.2 μm in diameter and contain a fine granular,
at a short distance from the central vein (outward zone III of the liver

2
J. Gilloteaux
homogeneous and osmiophilic content with either one or two distinct
crisscrossing, curved and densely contrasted crystalloids of uric acid
oxidase appearing together and intersecting as median line(s) (Fig. 1
D). Scattered throughout the hepatocyte cytoplasm a few auto-, het-
erophagosomes or residual (lysosomal) bodies with a few lipofuscin
bodies in the form of extremely electron densely contrasted structures
of irregular shape ranging from 0.2 to 0.8 μm in diameter can be seen.
Finally, a small number of spherical, fatty inclusions (0.5–1.5 μm diam.)
can be found in the hepatocytes facing the sinusoids typically lined by
their endothelial lining. The bile tract initiated as bile canaliculus by
the hepatocytes can also be detected in Fig. 1 A to D. There, the ca-
naliculi are typically surrounded by peroxisomes and lysosomes and
residual bodies (Fig. 1 D). The Syrian hamster canaliculi display a
narrow, circular-to oblong shaped lumen, about 0.5 μm in small dia-
meter, and they also can reach 2.5 μm in their longest diameter (Fig. 1
A-B and D). In all segments of the bile canaliculi, the adjacent hepa-
tocyte microvilli make the canaliculi lumen crowded and narrow for the
bile secretory products that will be moved by the hepatocyte sustaining
cytoskeletal rings. Often small membrane-like whorls or bleb-like
structures are observed. All the canaliculi are clearly delineated by the
junctional complexes of the forming, adjacent hepatocytes (Fig. 1A–D).
3.2. Intralobular to juxtalobular bile ductules or cholangioles or canals of
Hering
These channels are not always easily observed by electron micro-
scopy but, in some favorable fields of view, they locate between the
outermost lobular hepatocytes as narrow channels lined by flat elon-
gated to cuboidal cells with evident electron densely contrasted, epi-
thelial junctional complexes. They possess numerous, typical microvilli.
At low magnification and at first glimpse, no remarkable structure ap-
pears amongst some of the crowded surfaces and in the adjacent lumen.
However, using higher magnified views, one observed in some apical
surface as brandishing, a primary cilium can be found (Fig. 2 A and B).
Several short cross- and oblique sections of these cilia can be seen with
their content of microtubules. In these as well as in the other
Fig. 2. A-B: Intralobular cholangiole views at low (A) and high (B) magnifi-
cations depicting in its most central areas of the lumen oblique sections of 3
segments of undulating primary cilia indicated by arrows. Notice the junctional
complexes and cytoskeletal components in the cholangiocytes. Scales are 1 μm.
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Translational Research in Anatomy 19 (2020) 100063
Fig. 3. Hering canal or juxtalobular cholangiole lined by simple cuboidal cells
with apical, typical and blunted microvilli. Small, narrow lumen lined with
microvilli appear on both left and right of the main one corresponds to a
junction between three merging cholangioles. Notice the adjacent hepatocytes
(H) while interlobular connective tissue is shown on the upper and lower areas
of the micrograph. Scale is 5 μm.
observations described later, the cilia sections contain microtubules,
but they do not appear to have connections with a central doublet core
as they would be organized in typical cilia cross-sections. Fig. 3 illus-
trates a cross-section of an intralobular channel located in the stromal
tissue of the portal space adjacent to the lobular hepatocytes. This type
of duct is an extension of the canal of Hering. The lining epithelium of
this type of duct is constituted by 4–5 tightly sealed, cuboidal cho-
langiocytes and the resulting common lumen is very narrow. In addi-
tion, the epithelium of this type of tubular structure accommodates a
small lumen in the same field of the cross-section, as illustrated, and,
additionally, a smaller lumen in the opposite side, between the cuboidal
cholangiocytes, suggests that the entire tubular structure illustrated in
Fig. 3 is actually an example of tubular confluence of three cholangioles
or Hering ducts exiting the outermost zone of a liver lobule. The cho-
langiocytes are coated with short microvilli, and short apical bulges can
be seen. The cells are closely attached laterally and basolaterally by
prominent junctional complexes appearing as well-contrasted patches
between cell apices near their luminal side. In addition, several small
desmosomes can be seen distributed along the lateral surfaces, and
make additional intercellular, mechanical attachments. Nuclei are
spherical, centrally-located, and the narrow adjacent cell apices are
sealed by remarkable junctional complexes. They contain scattered,
small ovoid to round mitochondria, some smooth ER, rare fatty droplets
as minute inclusions with a few lipofuscin bodies. The cholangiocytes of
the duct of Hering are aligned along their evident basal lamina and
possess thickened basal membrane surfaces suggestive of a rich cell to
basal lamina connections with many specialized transmembranous
macromolecules (integrins, etc) and the external matrix is represented
by segments of small collagenic bundles sandwiched in the narrow
spaces intervening between these canal and other adjoining, matrical
spaces of the portal stroma containing some fibroblasts/fibrocytes and
their trailing extensions.
Large interlobular bile ducts (examples in Figs. 4 A – B, 5 and 6)
were found nearby peripheral regions of outermost lobular hepatocytes
and, thus, are better separated from them into the portal spaces by the
presence of more than one intervening structures: one or more delicate
matrix component (basement membrane and fibroblastic extensions)
with a more or less abundant matrix associated with other interlobular
J. Gilloteaux Translational Research in Anatomy 19 (2020) 100063

Fig. 5. Confluent interlobular cholangioles depicting luminal primary cilia.

Notice the intricate, entwined basolateral cholangiocyte regions and, in the
upper area, an histiocyte containing 3 centriolar bodies.Scale is 5 μm.
Insert: In a junction between two cholangioles, an encrypted cilium is found
near cell's junctions. Scale is 10 μm. Insert in view as attached. Scale is 1 μm.
Fig. 4. A: An oblique to cross-section view of a cholangiole (B) is illustrated
among the connective cells and tissue of a portal space where one cholangiocyte
bears a long, primary cilium; b: hepatic arteriole; L: lymphatic capillary; M:
macrophage; Pv: portal venule. Scale is 5 μm.
B: An enlarged view of the primary cilium viewed in A demonstrates its basal
body and rootlet reaching deep in the cytoplasm. One cross-section of another
primary cilium appears in the lumen above the primary cilium (arrow). Scale is
1 μm.

structure(s) such as a lymphatic capillary and adjacent to minor bile

ductules and stromal component of the portal space such as macro-
phages and other connective cells (Fig. 4 A). Specifically, Fig. 4 A de-
picts a cross-section of a small bile duct lined by a large number of
cholangiocytes, i.e in this case, at least seven or more cells. The surface
cholangiocytes show little number of microvilli and, when visualized,
they are short and even bifurcated (Fig. 4B) and the apical areas show
numerous small microsomal-like or endocytotic vesicles. In addition to
all the features described previously (cell junctions, organelles, etc), the
most intriguing structure demonstrated in this bile duct is the presence
of sections of a single, apical primary cilium in the epithelial cells
(Fig. 4A and B). There, the cilium can be viewed in its almost entire
length that does not appear to exceed more than 6 μm. In Figs. 5 and 6
and, better in its enlarged view of its insert, one notices in the ductal
lumen space another oblique section of an adjacent primary cilium. The
cilium demonstrated has no evident basal body and two perpendicular
Fig. 6. Example of cholangiocyte segregating part of its apical cytoplasm and
rootlets where the striation is about 40–60 nm in periodicity. The mi-
forming an excision piece (e); in the lumen of the Interlobular bile ductule two
crotubular components do not appear to forming doublets as in a ty- oblique sections of primary cilia (arrow) can be viewed. Scale is 1 µm.
pical cilium axoneme. Dark and pale cholangiocytes and at least two adjacent oval/intermediate cells
Another interlobular bile ductule is illustrated in Fig. 6 and insert can be viewed in the upper right (O). Microvilli are less prominent in the cells’
which lumen is associated to an adjacent, narrow lumen indicating a apices and number of ongoing, apical decapitations of the lining cholangiocytes
confluence of two bile ductules. At this level of the biliary tree, the are seen. A basal body of a primary cilium is arrowed. Scale is 5 µm.
epithelial cell lining is composed of low cuboidal cells with large sized,
interdigitated lateral and basolateral membranes displaying junctional
complexes. Each cholangiocyte possesses a large, oblong nucleus and a viewed as longitudinal and oblique sections of the bile space. It is
cytoplasm that contains several prominent polysomes, Golgi dictyo- among those microvilli that detect various sections of primary cilia as
somes, mutivesicular bodies and endosomes, and cytoskeletal filaments. indicated by arrows in Figs. 4–6. In the Fig. 6, the inserted micrograph
The apical surfaces are garnished with numerous short microvilli demonstrates that the convoluted cell surface encrypts or encloses a

4
J. Gilloteaux
primary cilium while in the narrow adjacent channel confluents with
the largest one and where a tall, primary cilium clearly appears in its
luminal space. In this example, most cholangiocytes shown whether cut
obliquely or not depict complex basal and basolateral surfaces with
intricate filopodia intermingling with each other adjacent cells while
maintaining a well-defined basal lamina separating the epithelium from
the subjacent lamina propria. There fibrocytes and their extensions
maintain the extracellular matrix, rich in collagenic fibrils, fibers and
bundles, as well as its associated ground substance. Fig. 5 also depicts
the surrounding matrix with a roaming macrophage that portrays
centriole body cross-sections.
In Fig. 6, a larger interlobular bile duct is shown (> 40 μm in dia-
meter) in the portal spaces of Kiernan and is lined with a simple cu-
boidal epithelium where the cholangiocytes demonstrate two main
morphologies: pale and dark cells. Some cholangiocytes are seen asso-
ciated with other adjacent, pale cells containing a high nucleus: cyto-
plasmic ratio and their large nuclei with branching nucleoli and poor
cytoplasm make them identified as oval or intermediate cells. These
duct cholangiocytes are surfaced with short but poorly distributed mi-
crovilli and a quick perusal of the fields of views allows us to detect
basal bodies along several of the lining apical surfaces of randomly
observed sections of pale and dark cells of this type of large ductule.
Numerous surface blebs can appear after being shed by self-excisions as
cell debris or fragments present in those luminal spaces suggest they are
bile materials, viewed with fuzzy profile can be contained in the lu-
minal spaces at the time of the organ harvesting (Figs. 5 and 6).
Moreover, numerous apical bulging apices exemplified by the micro-
graph illustrated in Fig. 5 with a cytoplasm apparently devoid of or-
ganelles but filled by ribonucleoproteins and glycogen, and ready to
excise itself in a process of self-excision similar to those described in
peculiar or autoschizic process [1,9,12,65–70]. Furthermore, and in-
terestingly, adjacent to the bulging apical aspect of the epithelial cell
illustrated in Fig. 5, it resembles the one detected in apocrine, secretory
function gland cell, at least two cross sections of primary cilia can be
noticed.
4. Discussion
4.1. The primary cilia
It is quite remarkable to also recall that if the primary cilium was
observed and described a long time ago [15], their detailed morphology
[59], but their physiologic functions have only been recently in-
vestigated in several types of epithelial cells or cells derived from em-
bryonic epithelial layers [18–48] that is why, from in vitro studies, so
many cell types have been found to bear a primary cilium because the
structure appeared and grew in mitotic cells and underwent resorption
during interphase or soon after going out of the cell cycle
[30,31,39,45,72,73]. Their relationship with the basal body-centro-
some [74–77] during the active cell cycle [45,71] made some authors to
suggest that, in addition to be a developmental mediator, the primary
cilia would be having a role in carcinogenesis and stem cell functions
[39,60,78–84]. If the speculated and some proven functions have been
noted in the literature, the long list of growth factors involved in the
primary cilium has been summarized [45,85] without any fine structure
comprehensive data or sheltered ‘cilia’ found in this study is still in need
to be understood, whether degenerated or growing features.
At first glance, with the transmission or scanning electron micro-
scopes, the primary cilia appear similar to the typical cilia as they
protrude and project out of the cell surface, usually apical in the case of
epithelial or glandular linings but they appear as stiff and straight or
bend, they also can be either shorter or much more slender and longer
than the classic cilia that are exclusively functioning to move materials
along cell and tissue surfaces. The primary cilia are known to lack a
central pair of microtubules usually needed for generating force, hence
are immotile and can be described as 9 + 0 pattern cilia and display a
5
Translational Research in Anatomy 19 (2020) 100063
progressive decrease in the number of their microtubular axonemal
component. If motility is the primary function for a cilium, the primary
cilia are immotile and are only involved in sensory function(s), as
shown long ago by the photoreceptors of the retinal epithelium
[57,79,80].
A plethora of reviews about the primary cilia that have appeared in
several biomedical journals [e.g. 29–48,59,60,71–81,
84–88,90,93,97–107] are certainly informative about the diverse tis-
sue's occurrence, but some of those, published with a few weeks apart,
often make information redundant and have not brought new mor-
phological information. In fact, there are reviews that make the topic
hard to be understood by the unprepared readers because did not dis-
tinguish between the typical, motile ‘cilium’ and ‘flagellum’ from ‘pri-
mary cilium’ or even classified them as one ‘immotile cilium’ even if
this cilium has never been motile… because it does not have central
microtubules as reviewed in Ref. [57] and recently brilliantly shown in
3-D views [59]. Finally, as it concerns the terminology, several pub-
lications on the primary cilium named it ‘organelle’ even though this
name should be only given to intracellular membrane-bound, sub-
cellular structure, i.e. nucleus, peroxisome, lysosome, mitochondrion,
instead of using the term ‘cell appendage’ (usually apical, in the epi-
thelial cells) as recommended [91].
4.1.1. Mechanosensors
Initially, it is Schwartz and others [92] who determined that fluid
shear bending the primary cilium can be used as mechanosensory de-
vice. A series of proteins of the cilia membrane and cytoplasmic support
molecules illustrate diverse integrated roles in various ciliary functions.
Mesothelial and endothelial cells display in vivo and in vitro apical,
primary cilia [93] and, in the endothelial cells used as study model
[94–96], it was noted that polycystin-1 (required for cilium function)
and polaris (required for cilium structure) are important mechan-
osensitive molecules. In the endocardial endothelium as ‘solitary cilia’
their function was confirmed as flow sensing ‘antennae’ in the em-
bryonic heart: after targeting those endothelial cells by colchicine or
stabilizing them with paclitaxel, a taxol derivative, the connections
(probably those authors implied the rootlet structure?) between the
cilium and the cytoplasm triggered a modification of the integrity in the
microtubular or other internal (cytoskeletal) junctions were essential to
sense and transduce the shear stress [94].
4.1.2. Modulators of cell differentiation
Primary cilia can be detected in actively mitotic cells and remained
in some cells that become specialized receptor appendages. The most
evident are the photoreceptors of the retina and in cells that retain some
specific transducing functions and mediating cell differentiation by
interacting with the extracellular matrix or able to receive differ-
entiating factors: i.e. chondroblast to chondrocytes [97–102]. In os-
teocytes the Indian hedgehog -activated smoothened receptor is a key
player along with a parathyroid-related protein in triggering en-
dochondral bone formation [103]. Furthermore, the peculiar mor-
phology of chondrocyte, isolated in their lacuna, have no part nor
network sensing capability similar to the direct, intercellular gap
junctions found in osteocytes, would ‘feel’ stress within its extracellular
matrix at the joints during postural and exertions through their primary
cilia which then would intervene in modulating dynamic gene expres-
sion for homeostatic and repair functions [103]. The aforementioned
data were verified and it was suggested that a better knowledge into the
osteocyte transduction mechanisms would eventually develop a new
therapeutic approach to combat bone loss caused by osteoporosis
[106,107].
4.1.3. In dysgenesis of the urinary tract
The epithelium of the collecting ducts in the kidneys is made of
cuboidal epithelial cells sensitive to vasopressin and are long known in
histology to possess a single, apical cilium. Those were also one of the
J. Gilloteaux
first tissue that initiated some refined physio-morphologic studies about
the primary cilium [29,40,41,108–113]. The ductal cells and en-
dothelial cell primary cilia membranes, such as of the collecting duct
cells also contain those similar mechanosensitive molecules polycystin-
1 and -2 [94,95,108]. Another report showed that the membrane of
primary cilium possesses several 7-span membrane receptors which are
regulated by a system of β-arrestin proteins [112,113], and with ser-
otonin [113], somatostatin [117] and angiotensin II receptors [118].
Furthermore, the smoothened receptors are to be endocytozed by the
action of a multimeric complex made of β-arrestins and kinesin motor
proteins that can be interfered or antagonized by Patch (12-span) re-
ceptor activation through an intricate coordination between Hedgehog
transcription factors and probably more others [82,83,113–116]. In
addition to the molecules brought up in the previous paragraph, the
superfamily of TRPV ion channels located in the primary cilia plas-
malemma [119,120]; they were further studied by Liedtke and colla-
borators and 6 types of those channels existing in mammals were de-
scribed [119,121,122]. Among them, TRPV1, 2 and 4 were found to
function with transduction of osmotic stimuli and to regulate osmotic
homeostasis from worm to mammals [123–126]. The specialized ion
channels can explain how their influence on some transducing proteins
could inform the cells about the extracellular microenvironment during
differentiation [108], for example, those able to interact with channels
and Ca 2+ signaling [45,50,83,109,127] and, at the end, can modify AQ
and other channels to control the homeostatic secretion of ions in re-
sponse to specific ligands affecting membrane cilia receptors. In the
case of the collecting ducts they allow body water retention as it would
be in the modification of bile composition by cholangiocytes. Macro-
molecular receptor studies through several specific developmental
transductions mechanisms or influences have been noted and reviewed
in normal collecting duct cells [41,42,74,127,128] and in pathologic
polycystic disease [108,128,129].; in that regard, Eggenschwiler and
others [88] reviewed several transduction mechanisms regarding mul-
tiple aspects of normal and abnormal developments. In the adult
kidney, however, a lack of primary cilia or defect can lead to several
pathologies as well as in the respiratory, urinary, adipose (Bardet-Beidl
syndrome [130,131], endocrine [74–79], neural tissues [90] as well as
behavioral [44].
5. The primary cilia in cholangiocytes: flow sensing and
osmosensors
Even though the cholangiocytes of the biliary tract modify the bile
made by the liver through absorption and generate at least 40% of
volume of the bile secreted [49–54], only little data have been collected
on the bile tract primary cilia [132–134]. On the opposite, cholangio-
cytes that belong to the pancreas exocrine appears to each have a pri-
mary cilium on their apical surface [135]. This appendage was in-
vestigated and found to be a mechanical, flow sensing device but also as
osmosensor [58] by altering their intracellular Ca 2+ and cyclic AMP.
Furthermore, they also showed that through the cation channel
(TRPV4) specialized in sensing fluid tonicity for not only bile flow but
also the regulation of secretion of bicarbonate in the bile [58,112,136].
Huang and collaborators [57] cleverly isolated by adhesion and strip-
ping dissection the primary cilia from cholangiocytes. This experi-
mental procedure allowed them to perform a refined functional and
molecular study that comforted the other data about the sensitivity of
the receptors and their transduction factors. There, the primary cilia
have been again confirmed not only able to detect changes in the lu-
minal flow but also to reveal tonicity as a result of transduction me-
chanisms involving cyclic AMP and TRVP4-Ca 2+ channels located in
the osmosensory membrane that regulate bicarbonate secretion
[50–52,58,132]. TRVP4 is expressed in several organs (kidney, trachea,
lung, brain, etc) on cells exposed to crucial osmotic changes. Primary
cilia have been found on A, B, and D cells of the islets of Langerhans
[136,137] wherein several of these islet cells found their origin in the
6
Translational Research in Anatomy 19 (2020) 100063
exocrine ductal cells (i.e. some ‘oval cells’ that can become cholangio-
cytes -see in paragraph 4 as confirmed by pathology [128,129] and
from in vitro and in vivo studies [138–143]. This growth and differ-
entiation of from the ductal cholangiocytes into B cells, for example,
can be achieved because in some tissues the primary cilia maintain
platelet-derived growth factor (PDGF) receptors which can, and with
appropriate external or internal stimulus, favor cellular responses that
include chemotaxis, proliferation or eventually carcinogenesis [129].
6. In the hepato-biliary tract of the Syrian hamster
The morphologic and functional characteristics of the gallbladder in
human and animals that possess a gallbladder are similar: the gall-
bladder is an accessory organ of the digestive tract that stores bile made
by the hepatic, exocrine secretion delivered by the bile tract ducts. It is
also remarkable to underline that the Syrian hamster liver histology [8]
and bile composition [64,144,145] also resemble that of human [129].
6.1. The biliary outflow tract ultrastructure
The hamster liver usually shows, like in human (contrarily to pig,
camel), a poorly developed connective tissue stroma, mainly evidenced
in the interlobular, portal spaces of Kiernan. Furthermore, the bile tract
of the Syrian hamster follows the classic morphology and pathway of
the human type [8]. Bile circulates outwardly from the liver portal
lobule plates via those channels made by facing grooves of hepatocytes
or bile canaliculi sealed by tight junctions [17]. Canaliculi are also
surrounded by hepatocyte cytoskeletal elements, namely actin fila-
ments [146–150] that can be altered by toxins and whose disturbance
produces cholestasis [151–153]. Adjacent to the canaliculi, as found in
other rodents, hamster hepatocytes harbor clusters of lysosomes as well
as peroxisomes [154–157] and the peroxisomes viewed here are typical
for Syrian hamster, characterized by their urate oxidase crystal shape
[157,159].
6.2. Bile ducts and gallbladder
The channels lined by squamous to low cuboidal cells at the edge of
the lobule, when distinct, are known as intralobular cholangioles (or
canals of Hering), continued by the same channels named juxtalobular
and interlobular bile ductules as lined by cuboidal cholangiocytes.
Finally, these confluent bile ductules into wider lumen interlobular bile
ducts lined by more cuboidal cells to bring the bile to reach the gall-
bladder via the cystic duct. In the gallbladder, the very diluted and
fluidic bile is modified and concentrated by removing water and some
electrolytes between meals. Bile is concentrated 8–10 times in the
human and in the Syrian hamster [158]. Other important functions are
also performed by the epithelial cells of the gallbladder and parts of the
bile tract, i.e. to secrete and excrete xenobiotic compounds and hor-
mones metabolized by the hepatocytes. The secretion of concentrated
bile with bicarbonate into the duodenum is initiated by hormonal re-
sponses to the arrival of fats and amino acids in the duodenum. Some of
the bile is reabsorbed in the intestines (the entero-hepatic circulation)
after affecting the uptake of lipid-soluble alimentary compounds. In
reference to those functions, the gallbladder is a reservoir and a me-
chanical pump [159]. The size, amount and quality of bile produced,
and extent to which bile is concentrated varies among species [160].
The gallbladder contractility is affected by cholecystokinin [161]
modifying the local neuronal pathways [162,163] while secretin and
other stimuli, controls the bicarbonate secretory functions of the cho-
langiocytes [132–135,164,165]. In addition, interstitial cells of Cajal
(ICCs), a.k.a. telocytes, that are present (but not studied here) in the
gall bladder wall and throughout the extrahepatic biliary tree can have
major implications not only for gallstone disease in regards of motility
but also for poorly understood conditions such as sphincter of Oddi
dysfunction and acalculous biliary dyskinesia [166–176]. Furthermore,
J. Gilloteaux
even though studies are only a handful [177–180], altered bile com-
position, such as supersaturated bile toxicity, in patients with chole-
lithiasis and, in most cases of acute inflammation or chronic defects of
the biliary tract, ICCs reduced number of the gallbladder and extra-
hepatic, biliary duct walls would reduce their motility [181,182], due
to either innervating and sensing dysfunctions [10,183–185] and/or
metabolic along with transduction controls perturbed, as consequences
of the supersaturated bile toxicity. It is interesting to note that some
other bile components (ω-3 PUFA, glycocholic and taurocholic acids)
may exert protective effects on ICCs. In this case, ω-3 PUFA would
represent a possible option to prevent formation of gallstones, impeding
cholesterol initial depositions [186].
Inferring from other data, discussed in the following paragraph, the
primary cilia located along the bile tract can act as antennae or flow
rate and, based on the current literature, can be osmo-sensors for the
secretion of bicarbonate by the cholangiocytes. Finally, the production
of a protective film of mucus at the apical surfaces of the gallbladder
and of the tract is a reminder that the tract derived from the foregut,
endoderm lining. Some of the mucus protective film can also originate
from the cystic duct mucosa containing some small simple, branched
acinar and intraepithelial mucus glands in hamster and human
(Gilloteaux, unpublished). In some animals, such as rat and deer as well
as some birds (e.g. pigeon) that eat almost continuously, they have no
gallbladder and the constant flow of bile from the liver to the intestine
is probably adequate owing their chronic digestive craving and, thus
feeding habits [187,188].
6.3. c The primary cilia of bile ducts in vivo and in vitro
It is now clear from this study that the cholangiocytes of the in-
tralobular and extralobular bile ductules (canals of Hering or cho-
langioles) can be surfaced with a single, primary cilium while in vitro
cultivation and morphologic evaluation of intrahepatic bile duct cho-
langiocytes have not yield any primary cilium, instead none or 20–30
cilia per cell [189]. It is also interesting to note that from our data, these
cell appendages were present on the cholangiocytes of the smallest
diameter ducts. Only a handful of reports have studied with electron
microscopy the Hering canals or cholangioles of mammals. In the de-
veloping human bile tract of a 320 mm human fetus illustrated one
‘cilium’ in one of the Hering canals (or juxtalobular and interbiliary
ductules) [17]. It is not clear from our study what would be the
minimum diameter to find these cholangiocyte appendages but, like
shown in the human liver, their presence detected in very narrow
Hering canals of the hamster. These channels and larger ones are the
one receiving the flow of bile of the lobular bile canaliculi and to ‘sense’
whether the flow characteristics and quality.
In vitro, the bile cholangiocytes located at the edges of hepatic lo-
bules formed bile ducts, become surfaced by typical microvilli along
with prominent, junctional complexes as shown in our data. The
narrow, layer of flat to cuboidal cholangiocytes, initiating the wall of a
channel away from the liver hepatocyte bile canaliculus, can have rapid
turnover, more rapid after ischemia [157,191,192] and similar to those
found in human tissues) with signaling capabilities [194,195], grow
aquaporin (AQ) channels [62,196–198]. In some case, ‘single cilia’ were
noticed [193,194]. Only bile ducts larger than 15 μm in diameter are
regulated by secretin hormone actions [164,199]. The same secretin
favors an increased bile flow as well as the apical insertion of AQ-1
channels [173–175] and there are now at least 7 out of 13 types of AQ
channels known in the liver, gallbladder and pancreas but AQ1 is the
main one in the biliary tract [198]. Bile acid feeding increases cho-
langiocyte proliferation and ductal secretion [164,199,200]. Antag-
onistically, somatostatin seems to inhibit the secretin action on cho-
langiocytes by decreasing their secretory functions while activating bile
secretion [136,200]. Following these previous remarks, it is surprising
7
Translational Research in Anatomy 19 (2020) 100063
that we did find long, wavy primary cilia in the intralobular and in-
terlobular cholangioles, considering the small diameter of the bile
channel. The micrograph displayed in Fig. 4 remind the 3-D micro-
graphs and reconstructed views reported by Sun's group [59], along
with the tiny cross section found in the same lumen. Based on the
Hagen-Poiseuille law, these appendages of the surface epithelium
would act sensors or antennae for flow and quality are adequate
[201–203] because located in small to wide duct where the gradient of
friction or ‘resistance’ would be highest near the wall [204]. Some flow
rate alteration such as impeded, in cholestasis or of altered osmotic
pressure linked to bicarbonate modification would then be detected
along the ducts, closest to the liver production bile site. This potent
feedback response as such, would need further experimental and
translational clinical clarifications, already evoked by several reports
[48–58,133–136,205]. In the same Fig. 4, the primary cilium rootlet,
part of the stability component of the primary cilium, is viewed with its
typical striation [205]. This structure evidences the linkage between the
cilium and other cytoplasmic structures where the regulatory and
translational macromolecules can perform their functions in order, for
example, modify AQ1 channels and bicarbonate-dependent channels to
control the quality of bile secreted [199–203]. The largest bile ducts
observed in the Syrian hamster showed populations of pale and dark
cells, which remind those noted in Refs. [164,165,177]; those mor-
phologic differences were similar to the ultrastructure revealed in Refs.
[148,149] in the common bile duct of other rodents.
7. The oval or intermediate cells
Comprehensive surveys dealing with the development and differ-
entiation of the entire biliary outflow structures out of the hepatic di-
verticulum in human and experimental models included those data
reminding us that the intralobular bile ducts contain liver reserve ‘stem’
cells that could be stimulated through factors influenced by adjacent
damaged, connective tissue and the possible actions of multiple specific
factors reviewed out of rodent and human components [159,206–211].
The so-called oval cells [89,213–215], also called “intermediate cells”
[206,207], can appear more difficult to reveal simply by morphology,
especially having new tools of molecular markers and ancillary tech-
niques. Even though, transitional cells or intermediate cells as ‘stem
cells’ type are still difficult to simply reveal them with morphology,
unless using molecular markers and other ancillary techniques. Even
though, ambiguous ‘intermediate cells’ as ‘stem cells’ type is still diffi-
cult to uncover with transmission electron microscopy alone, as re-
marked, as they exist between liver cells associated with the initial
segment of the biliary duct cells [164,215]. Even though other authors
did not find these ‘intermediate cells’ in normal and pathologic mate-
rials [17,33,34], Picardi and others [217] noted them with electron
microscopy that intermediate, cholangiocyte stem cells exist in the
human fetus similarly of the data presented in Ref. [18].
Various transitional cells appear between biliary ductule cells and
liver cells and are likely issued from the cholangiocytes [150,212–214].
These oval (intermediate) cells that can be found associated with or
closely adjacent to the cholangiocytes could also suggest the primary
cilia to act as ‘antennae’ signaling these oval cells to trigger proteome
expression of differentiation into either cholangiocytes or hepatocytes
[88,194,195]. Such potential reactive ‘ductular’ activation was ob-
served in human liver defects [150,199,202,203,212,214,216] or in in
vitro experiments [210,211,215], as well as in old and very recent
observations in murine or human hepato-biliary-pancreatic develop-
ment [34,37,142,164,215] where more data have established the pri-
mary cilium would grow as a signal detector of transduction with the
already noted factors, including PDGF, thus a defective signal would
trigger or be causing a cholangiopathy [50–52,62,150] like other dys-
geneses found for the kidney [53,54].
J. Gilloteaux Translational Research in Anatomy 19 (2020) 100063

8. Conclusion hamster: evidence for an intramucinous nucleating process and downregulation of

This morphologic report comforts data obtained in rats where the [14]
primary cilium in bile channel cholangiocytes have been studied and
reviewed in light of rat biliary tract pathophysiology
[194,198,217,218]. Those data and ours found in the Syrian hamster [15]
can be relevant to human liver functions and should motivate further
translational, developmental and clinical investigations to further [16]
clarify those related with the pathophysiology of the biliary tract as [17]
they involve not only the control of low sensing (i.e. mechanosensing)
similar to those shown in Ref. [92], chemo-as well as osmosensory [18]
[38,49,52] and/or affecting normal and abnormal developmental as-
[19]
pects of the bile duct system [58,150,210,212,213,216–218].
Ethical statement [20]
[21]
The ethical statement is in the material and methods paragraph.
[22]
Declaration of competing interest
[23]
The author declares that he has no known competing financial in-
[24]
terests or personal relationships that could have appeared to influence
the work reported in this paper.
[25]
Acknowledgements
[26]
The author thanks the Summa Research Foundation and the late
Thomas R. Kelly M.D. fund, Department of Surgery, of the Summa
[27]
Research Foundation, Akron Ohio, financially assisting in this research
topic between 1985 and 1995. Data were partly supported by the [28]
Fondation Nationale de la Recherche Scientifique de Belgique (with Dr [29]
J.-J. Vanderhaeghen, ULB) during a summer leave of 1993. Mr. S. [30]
Getch, Summa Communication Specialist, is recognized for his assis- [31]
tance for imagery. Manuscript was completed during the author's te-
[32]
nure at St George's University International School of Medicine in
Newcastle upon Tyne, United Kingdom.
[33]
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