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Microorganisms commonly use efficient motile 9 + 2
cilia to move around1–3. These cilia also cover numerous
epithelia in vertebrates, in which they drive the move-
ment of extracellular particles or generate fluid flow.
These motile cilia are organized around an axoneme
consisting of nine microtubule doublets arranged in a
circle around two singlet microtubules4. The formation
of an axoneme is nucleated by a basal body, which is
composed of triplets of microtubules in a nine-fold sym-
metrical arrangement (related to the centrioles found
in the centrosome) that is docked at the plasma mem-
brane5. Dynein ATPase motor proteins cause adjacent
axonemal microtubules to slide over each other to bend
the cilium. Each beat of a cilium consists of a fast effec-
tive stroke that propels fluids and a slow, curved, recov-
ery stroke against the flow of the fluid that brings the
cilium back to its original location4. The central pair of
microtubules is suggested to be necessary for this planar
stereotypical waveform of ciliary beating6.
Generation of cilium-powered directional fluid flow
at the tissue level requires a dense array of cilia beating
unidirectionally at a high frequency (5–35 Hz). The term
multiciliated cell commonly refers to a cell that extends
multiple 9 + 2 cilia from its apical surface. In mammals,
multiciliated cells cover the lumen of the ear–nose–
throat sphere, the lungs, the brain ventricles and part
of the female and male reproductive tracts. Each multi-
ciliated cell nucleates from 30 to 300 cilia, depending
on the organ. Multiciliated precursors with only one
centrosome must therefore amplify up to 300 cen-
trioles to support cilia nucleation7–16. This amplification
occurs through the formation of intermediate structures,
deuterosomes, which are composed of centrosome-
related elements. Deuterosomes support massive cen-
triole formation downstream of a molecular cascade that
is shared with the cell cycle centrosome duplication pro-
gramme17–20. Once docked at the apical plasma membrane,
the new centrioles become basal bodies and acquire a
rotational polarity that determines the direction of planar
cilia beating. Basal bodies are distributed evenly across
the apical membrane in the airways and reproductive
organs. In the brain, however, basal bodies are clustered
towards the downstream flow of cerebrospinal fluid
(CSF), thereby defining its translational polarity21. Ventricle
epithelia are composed mainly of multiciliated cells in the
brain, whereas in the airways and oviducts, multiciliated
cells are interspersed with epithelial secretory cells.
Abnormalities in centriole formation or migration,
the orientation of cilia beating or its frequency result in
pathological conditions. The massive cellular changes
taking place in differentiating multiciliated cells have
been documented by extensive electron microscopy
studies. Recently, there has been renewed interest in
multiciliated cells owing to the development of ‘omics’
methodologies and progress in live and super-resolution
microscopy. In this Review, we provide a cell-to-organ
overview of a century of data on multiciliated cells in
verte brates. We discuss multiciliated cell fate determin-
ants and describe both the dynamics and molecular
events at play during the differentiation of a cell contain-
ing one single centrosome into a polarized cell with tens
of centriolar structures nucleating a functional motile
ciliary tuft. Last, we exhaustively discuss the locations
and functions of multiciliated cells in mammals,
École Normale Supérieure,
PSL Research University,
CNRS, Inserm, Institut de
Biologie de l’Ecole Normale
Supérieure (IBENS),
F-75005Paris, France.
nathalie.spassky@ens.fr;
alice.meunier@ens.fr
doi:10.1038/nrm.2017.21
Published online 12 Apr 2017
Apical plasma membrane
In epithelial cells, the
membrane located towards the
lumen in a body tube or cavity.
Rotational polarity
The orientation of ciliary
beating.
Tr a n s l a t i o n a l p o l a r i t y
Planar asymmetric localization
of clusters of basal bodies
onthe apical area of
multiciliated cells.
Ven tri cle ep ith eli a
Single layers of cells lining the
surface of the brain ventricles.
Secretory cells
Non-ciliated cells that secrete
mucus into the respiratory
tract, or fluids rich in nutrients
into the reproductive tracts.
The development and functions
ofmulticiliated epithelia
Nathalie Spassky and Alice Meunier
Abstract | Multiciliated cells are epithelial cells that are in contact with bodily fluids and are
required for the proper function of major organs including the brain, the respiratory system and the
reproductive tracts. Their multiple motile cilia beat unidirectionally to remove particles of external
origin from their surface and/or drive cells or fluids into the lumen of the organs. Multiciliated cells
in the brain are produced once, almost exclusively during embryonic development, whereas in
respiratory tracts and oviducts they regenerate throughout life. In this Review, we provide a
cell-to-organ overview of multiciliated cells and highlight recent studies that have greatly
increased our understanding of the mechanisms driving the development andfunction of these
cells in vertebrates. We discuss cell fate determination and differentiation of multiciliated cells,
andprovide a comprehensive account of their locations and functions inmammals.
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Basal cell
Undifferentiated airway cells
localized deep in the
respiratory epithelium
andcharacterized by the
expre ssion o f p63 an d SOX2.
Coiled-coil domain
α-Helix-containing structural
domain in proteins that are
coiled together to form dimers
or trimers.
andhighlight atypical and understudied multiciliated
cells that would be of interest for further studies.
Multiciliated cell fate determination
Multiciliated cells are either mostly produced during
development and exist for years (in the brain) or con-
tinuously regenerated throughout life (in the airways
and reproductive organs). Identification of the progen-
itors of multiciliated cells is a first step towards under-
standing the mechanisms that direct multiciliated cell
fate determination. Recent studies have shown that in
different organs, multiciliogenesis shares common fate
determination mechanisms.
The progenitors of multiciliated cells. In the airways,
uterus and oviducts, multiciliated cells are regener-
ated throughout life22–24. In the airways, multiciliated
cells originate from p63 (also known as TP63)- and
SOX2-expressing progenitors. p63 and SOX2 are
transcription factors that are highly expressed in the
basal cell lineage and are required for the development
and regeneration of ciliated cells25–27. During post-
natal growth and adulthood, multiciliated cells derive
from basal cells or secretory cells, depending on tissue
identity and cellularcontext28.
In the adult mammalian brain, multiciliated ependy-
mal cells form a continuous layer that lines the walls of
the lateral, third and fourth ventricles. These cells are
formed only once during development16; their replace-
ment during ageing or after injury is minimal29,30. Cell-
fate tracing experiments demonstrated that radial glia
are the progenitors of ependymal cells in the mouse
brain16. Although radial glia are heterogeneous and
show restricted potential to produce various subtypes
of neurons in some parts of the brain31, no regional dif-
ferences among multiciliated progenitor cells have yet
been identified.
The progenitors in other multiciliated organs are yet
to be characterized.
Cell fate determination. It is likely that multiple inter-
cellular signalling pathways cooperate to specify the
transformation of progenitor cells into multiciliated
cell precursors. In many epithelial tissues, this choice
seems to be the default fate for progenitor cells. In the
zebrafish pronephros, mammalian airways and embry-
onic epidermis of the amphibian Xenopus laevis, the
inhibition of Notch signalling controls the balance
between the formation of ciliated cells and other cell
types32–38. Activation of the Notch pathway by genetic
and pharmacological approaches silences ciliation in
cells, which then continue to expand and differentiate
into secretory cells. Conversely, inhibition of Notch
signalling leads to differentiation into ciliated cells at
the expense of secretory cells34,35,37–41. In X.laevis epi-
dermis and human airways, the microRNA miR-449
promotes multiciliogenesis by directly repressing
the Notch 1 pathway, which is induced by its ligand
Delta-like protein1 (REF.33) (FIG.1). These regulatory
processes are maintained in adult airways, as ciliated
cells are produced directly from basal (progenitor) cells
expressing p63 or indirectly from Notch 2-expressing
secretory cells42–45. The Notch ligand is provided directly
by the basal cells to maintain the pool of their daughter
Notch 2-expressing secretory cells, which otherwise
are converted into ciliated cells, as shown by the inhib-
ition of the Notch ligands jagged 1 (JAG1) and JAG2
(REFS45,46). Although the upstream signals still need
to be identified, similar inhibition of the Notch pathway
is required to arrest the proliferation of progenitor cells
and to specify their differentiation into multiciliated
cells in the brain and oviducts23,47.
The bone morphogenetic protein (BMP) and Notch
pathways might interact to control cell fate, asBMP
ligands inhibit multiciliated cell specification in the
X.laevis epidermis and in mammalian airways and
embryonic stem cells48–50. Conversely, the transforming
growth factor-β (TGFβ) regulators SMAD6 (also known
as MADH6) and SMAD7 (also known as MADH7)
promote the formation of multiciliated cells from
embryonic stem cells51.
The most-upstream activators of the multiciliated
cell transcriptional programme found so far are gem-
inin coiled-coil domain-containing protein 1 (GEMC1)
and multicilin (FIG.1). These proteins have a central
coiled-coil domain and were originally identified as
regulators of DNA synthesis52,53. Multicilin can inter-
act with geminin and inhibit its function as a negative
regulator of DNA replication54, and GEMC1 interacts
with CDC45 and DNA topoisomerase 2-binding pro-
tein 1 to promote DNA replication52. It is currently
unknown whether, as regulators of DNA replication,
they are directly involved in multiciliogenesis and,
ifso, how. GEMC1 and multicilin seem to initiate simi-
lar transcriptional programmes that are required for
multiciliated cell differentiation36,55–57. Notch inhibition
leads to the activation of multicilin, which is required
for the formation of multiciliated cells in the X.laevis
Figure 1 | Mammalian multiciliated cell fate determination. Notch 1-induced
proliferation of progenitor cells is inhibited in human airways by the microRNAs miR-34
and miR-449. Notch 1 inhibition leads to the activation of the master regulators of
multiciliogenesis, geminin coiled-coil domain-containing protein 1 (GEMC1) and
multicilin, which, together with the transcription factors E2F4 or E2F5, commit
progenitor cells towards multiciliation and induce differentiation through activation of
p73 and the motile ciliogenic pathway (MYB, forkhead box protein J1 (FOXJ1), regulatory
factor X 2 (RFX2) and RFX3), which triggers basal body amplification, active remodelling
of the cytoskeleton and ciliogenesis. EDM, E2F4– or E2F5–DP1–multicilin.
Proliferation of progenitor cells
Multiciliated cell precursor
Motile
ciliogenic
pathway
Multicilin,
E2F4, E2F5,
DP1 (EDM complex)
Notch1
GEMC1
miR-34, miR-449
(in human airways)
p73
FOXJ1
RFX3
RFX2
MYB
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Procentriole
Immature centriole or basal
body in the process of growth.
epidermis and kidney, and in mouse and human air-
ways36,58,59. The expression of GEMC1 precedes that
of multicilin, and both GEMC1 and multicilin con-
trol the generation of multiciliated cells in the mouse
brain47. GEMC1 is required for multicilin expression in
zebrafish and X.laevis60. The trachea and oviducts of
GEMC1-knockout mice show highly reduced expres-
sion of multicilin and genes required for centriole
biogenesis56, suggesting that GEMC1 acts upstream
of multicilin. Mice lacking GEMC1 show impaired
growth, develop hydrocephalus and are infertile.
The trachea, oviducts and brain of these mice lack
multiciliatedcells56,57.
Multicilin promotes the transcription of key genes
that are required for multiciliogenesis through inter-
actions with the E2F4 or E2F5 transcription factors
and their cofactor, DP1 (the E2F4 or E2F5–DP1–
multicilin ternary complex is referred to as the EDM
complex)55 (FIG.1). Although the interaction of GEMC1
with E2F5–DPI is controversial56,60, GEMC1 activity is
enhanced by E2F5 and is required to activate E2F5
target genes in tissues that give rise to multiciliated
cells55,43. Loss of E2F4 and E2F5 in mice impairs the
development of multiciliated cells in the airways and
the efferent ducts61,62. Interestingly, the multicilin gene
is located on chromosome 5q11.2 in humans and
chromo some 13D2.2 in mice; both loci harbour other
key regulators of multi ciliated cell formation, includ-
ing cyclinO (CCNO), CDC20B and mir-449a, mir-449b
and mir-449c33,58,63,64.
Multiciliated cell differentiation
In the mammalian brain, progenitor cells lose their
radial glial morphology and molecular characteris-
tics, and acquire multiciliated ependymal cell features
(expressing SIX3, CD24 and S100β) and a cuboidal
morphology16,65,66. This tr ansformation requ ires
active remodelling of the cytoskeleton and the local-
ization of tight junction proteins such as N-cadherin
on the cell membrane through the adaptor protein
ankyrin3 and the Hippo signalling pathway effector
Ye s - a s s o c i a t e d p r o t e i n ( YA P ) 67,68. In the airways and
in X.laevis epidermis, BMP signalling, the small Rho
GTPase RHOA and actin-based cell-autonomous activ-
ity dependent on the actin elongator formin1 contrib-
ute to the apical migration of basal cells to the outer
layer, where they intercalate among other cells and
resumedifferentiation27,48,69,70.
Massive production of centrioles. Mu lticiliated cell pre-
cursors, which contain one centrosome with a mother
and a daughter centriole, produce from 30 to 300 new
centrioles during the course of their differentiation.
These centrioles provide templates for the subsequent
growth of the corresponding number of motilecilia.
During canonical centrosome duplication in divid-
ing progenitor cells, mother and daughter centrioles
guide the formation of only one new centriole on their
walls. Centrosomal protein of 63 kDa (CEP63) recruits
CEP152, and they form a complex on each centrosomal
centriole. This complex initiates centriole biogenesis
through the activation of Polo-like kinase4 (PLK4)
and the subsequent stabilization of spindle assembly
abnormal protein 6 homologue (SAS6), which acts as
a scaffold for centriole formation71–73. During multi-
ciliated cell differentiation, this canonical duplication
programme is diverted to allow massive centriole
production. In response to expression of the EDM
complex, a paralogue of CEP63, deuterosome assem-
bly protein1 (DEUP1), is expressed55. DEUP1 can
oligomerize and form spherical electron-dense aggre-
gates called deuterosomes19. DEUP1 is also able to
recruit CEP152 and trigger the canonical centrosome
duplication cascade that drives centriole biogenesis19.
In parallel to DEUP1 expression, the components of
this cascade (including CEP152, PLK4 and SAS6) are
massively upregulated during multiciliated cell precur-
sor differentiation18,19,55,59,74–76. These elements organize
centriole amplification in multiciliated cells through:
first, a CEP63-dependent pathway that leads to the
bio genesis of centrioles directly from centrosomal
centrioles and, second, a DEUP1-dependent pathway
that leads to centriole biogenesis from deuterosome
structures19 (FIG.2). Consistent with the overall parallel
with the centriole duplication programme in cycling
cells, deuterosomes form on a centrosomal centriole
in the chick trachea and in the mouse brain14,20. In the
mouse brain, live imaging, super-resolution imag-
ing and electron microscopy show that the deutero-
somes are nucleated from the daughter centriole of the
centro some, suggesting the existence of a local micro-
environment that is conducive to the formation of
these auxiliary centrosome structures20. Whether this
immature, pre-existing centriole transmits informa-
tion to the deuterosomes77 or only provides a scaffold78
isunknown.
Centriole amplification is proposed to be organized
around three consecutive phases (FIG.2). The first is an
amplification phase during which several procentriole-
bearing deuterosomes sequentially form from the prox-
imal wall of the centrosomal daughter centriole. During
this phase, procentrioles are also growing directly from
each centrosomal centriole20. The second, a growth
phase, begins when all the deuterosomes havebeen
produced and the final number of centrioles has
beenreached; during this phase, all of the procentrioles
widen and elongate from centrosome and deuterosome
platforms14,20. The third is the disengagement phase,
during which all of the centrioles detach simultane-
ously from their centrosome and deuterosome plat-
forms to migrate to the apical membrane and nucleate
motile cilia14,19,20,79. Deuterosomes disappear at this stage
through an unknown process80, and the fate of the two
centrosomal centrioles remains unknown.
Interestingly, in the chick trachea, after a first round
of deuterosome-mediated centriole amplification from
the centrosome14, several small deuterosomes are addi-
tionally nucleated from newly formed basal bodies that
are migrating to the apical membrane15. This suggests
that additional sites and timing of deuterosome nucle-
ation can exist, maybe to increase or adjust the number
of centrioles produced.
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In addition to the multicilin–E2F–DEUP1 molecu-
lar cascade and the upregulation of common centro-
some duplication factors, the amplif ication phase
requires the activity of CCNO, which is a cyclin-like
prote in involved in deuterosome formati on and
function through an as yet undetermined mech-
anism76. People with pathogenic mutations in CCNO
have multiciliated cells with a reduced number of
centrioles and cilia81–83. In addition, the centriole-
associated protein coiled-coil domain-containing pro-
tein 78 (CCDC78) is required, together with DEUP1,
for CEP152 recruitment by the deuterosome and
subsequent procentriole formation18.
In contrast to the amplification process, which is
beginning to be understood, the signal that stops cen-
triole amplification and therefore regulates centriole
number is completely unknown. It seems to be corre-
lated with the onset of latent procentriole growth14,20,
suggesting that the two processes may be causally
linked. The signal that triggers the synchronous release
of newly formed centrioles by both centrosomal
centrioles and deuterosomes is unknown.
1
2 3
Centriole amplification
Centriole growth
Mother centriole
Centrosomal
mother
centriole
Centrosomal
daughter
centriole
Daughter centriole
DEUP1
CEP63
CEP152
PLK4
POC5
CP110
Centrin
Glutamylated
microtubules
CEP164SAS6–STIL
Deuterosome
in formation
Procentriole Completed
deuterosome
Centriole disengagement and docking,
and cilium formation
b
a
Daughter centriole
Multiple
rounds
Centrosome
100 nm
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Morphants
Organisms treated with
amorpholino oligomer
todecreas e gene expre ssion.
Distal appendages
Acces sory co mponen ts of
mature centrioles that are
involved in centriole docking
atthe vesicular or apical
plasma membrane.
Secretory vesicles
Cytoplasmic vesicles that
fusewith the apical surface
ofthecell.
Exocyst
A multiple-subunit complex
that is required for the
interaction of secretory vesicles
with the plasma membrane,
inpreparation for
membranefusion.
Tr a n s i t i o n z o n e
The most proximal region of
acilium, upstream of the distal
appendages of the basal body.
Centriole docking to the plasma membrane and cilium
growth. Following collective enmasse migration to the
apex of the cell, centrioles dock at the plasma membrane,
where they become basal bodies and nucleate motile
cilia. Centriole migration is actomyosin dependent84,85.
In mice, subsequent basal body anchoring requires the
assembly of an apical actin meshwork, controlled by
the actin regulators RHOA, ezrin, RAS and filaminA,
that develops downstream of the multiciliogenesis
factors miR-34, miR-449 and forkhead box proteinJ1
(FOXJ1)69,86,87. The network implicated in basal body
connection to the actin cytoskeleton has been thoroughly
investigated in the X.laevis skin model. It involves inter-
connected apical and subapical actin plates as well as
focal adhesion proteins79,88–90. Components of the planar
cell polarity (PCP) — that is, the coordinated polariza-
tion and alignment of cells — include dishevelled (DVL)
and the effectors inturned and fuzzy. These proteins also
regulate basal body docking through apical actin enrich-
ment, membrane trafficking or the interaction of basal
bodies with the cytoskeleton90–95. In addition, ciliation is
abnormal in mice with mutations in the PCP protein cad-
herin EGF LAG seven-pass G-type receptor 2 (CELSR2)
owing to defects in basal body docking96; thus, basal
body docking and polarity are, at least in part, controlled
by common regulators. Although basal body docking
does not seem to be defective in mice in which one of
the core PCP genes, vang-like 2 (Vangl 2 ), is mutated80,97,
undocked basal bodies98 have been observed in X.laevis
vangl2 morphants, suggesting that the mechanisms differ
in the X.laevis and mousemodels.
The appearance of vesicles bound to migrating basal
bodies in multiciliated cells9,10,84,99 sug gests that, a s in pri-
mary ciliated cells100, the formation of ciliary vesicles at
distal appendages during the early steps of ciliogenesis
facilitates basal body docking. Similarly, in X.laevis, Dvl
proteins were proposed to be involved in the recruitment
of secretory vesicles to basal bodies, suggesting that the
exocys t may participate in basal body migration, planar
positioning or docking to the plasma membrane92.
Inmouse airway multiciliated cells, the association of the
basal body component chibby with the distal append-
age protein CEP164 was shown to lead to the assembly
of ciliary vesicles mediated by the small GTPase RAB8
(REF.101). As in primary ciliogenesis100, the knock-
down of centrosomal protein of 110 kDa (CP110; also
known as CCP110), which is a distal centriolar protein
that controls centriole length, is required for axonemal
extension. Inmult iciliated cells, CP110 depletion is
mediated, at least in part, by the activity of miR-34 and
miR-449(REF.63).
In addition to its involvement in basal body docking,
FOXJ1 is a master regulator of the motile cilium tran-
scriptional programme. In cooperation with the regula-
tory factor X (RFX) family of transcription factors that
drive the expression of core cilium genes, FOXJ1 con-
trols the expression of motility genes102–109. Both RFX2
and FOXJ1 expression can be induced by multicilin55.
Inaddition, FOXJ1 can be activated directly by other core
components of the multiciliated cell differentiation pro-
gramme, such as GEMC1 and MYB56,57,59 (FIG.1). In paral-
lel with the FOXJ1–RFX network, TGFβ was recently
shown to be involved in assembly of both primary cilia
and multicilia, probably by controlling the expression of
transition zone prote ins110. As for primary cilia, the assem-
bly and maintenance of motile cilia in multi ciliated cells
rely on the intraflagellar transport machinery that carries
ciliary components along axoneme microtubules111.
Incontrast to the peripheral microtubule doublets of
the axoneme that are templated on centriolar micro-
tubules, the central doublet, which arises from the top
of the motile cilia transition zone and is required for
different aspects of cilia motility6, does not attach to any
recognizable structure. The nucleating mechanism and
the transcriptional components that drive the growth of
this central doublet are undetermined6.
Recently, the p53 homologue p73 was discovered
to be a major regulator of centriole docking and cili-
ation112–114 that lies downstream of the multicilin–E2F
network (FIG.1). Chromatin immunoprecipitation and
sequencing analysis predicted it to bind to more than
100 cilium- related genes113. Functional studies have
shown that it directs FOXJ1, RFX3 and miR-34 expres-
sion, which explains, at least in part, the docking and cili-
ation defects of p73 mutants112,113. p73 genomic binding
sites are also found in proximity to regulators of cen triole
amplification such as MYB113 but, according to func-
tional studies, p73 does not have an important role in
centrioleformation112,114.
Figure 2 | Mammalian multiciliated cell differentiation. a|Model for centriole
biogenesis in multiciliated cells. Centriole amplification (step1). About 10% of the
centrioles from which the multiple cilia will grow form in a deuterosome-independent
pathway that is initiated by centrosomal protein of 63 kDa (CEP63), whereas a
deuterosome-
dependent pathway initiated by deuterosome assembly protein 1 (DEUP1)
is responsible for the formation of around 90% of centrioles. Both pathways are active
concurrently and involve the same downstream molecular cascade, which is also shared
by the centriole duplication process in cycling cells. The CEP63-dependent pathway is
used by both centrosomal centrioles, whereas the DEUP1-dependent pathway is used
only by the centrosomal daughter centriole. During deuterosome formation,
procentrioles remain latent at an early stage of their biogenesis. Centriole growth
(step2). When the final number of centrioles is reached after several rounds of
deuterosome formation, the deuterosome formation process stops and all the latent
procentrioles grow and mature simultaneously from both centrosomal centrioles and
deuterosomes. At the molecular level, procentrioles become positive for the marker of
the late step of centriole assembly, protein of centriole 5 (POC5), and have their tubulin
polyglutamylated. Centriole disengagement and docking, and cilium formation (step3).
Centrioles disengage synchronously from the platforms on which they grow and migrate
collectively to the apical membrane. By this time, they have recruited CEP164, which
enables them to anchor on the membrane and nucleate the ciliary tuft. b|Electron
microscopy images show that both pathways of centriole biogenesis are active
concurrently. Accumulation of electron-dense material (composed at least of DEUP1
andthe deuterosome-associated protein coiled-coil domain-containing protein78
(CCDC78)) on the proximal side (that is, the lower part with respect to centriole docking)
of the centrosomal daughter centriole leads to the progressive formation of
deuterosome spheres (arrows). These spheres then extend small latent procentrioles
(arrowheads). Concomitantly, procentrioles grow directly from the proximal part of both
centrosomal centrioles (third panel from the left; note that only the daughter centriole
isshown here). Whereas procentrioles that grow directly from the two centrosomal
centrioles do not disengage, deuterosomes detach from the centriolar wall and
accumulate in the cytoplasm, allowing another round of deuterosome formation.
Formation of up to two deuterosomes can be observed at the same time on the daughter
centriole. CP110, centrosomal protein of 110 kDa; SAS6, spindle assembly abnormal
protein 6 homologue; STIL, SCL-interrupting locus protein. Part b is adapted with
permission from REF.20, Macmillan Publishers Ltd.
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Microtubule organizing
centre
Major site of microtubule
nucleation and anchoring
in a cell.
Basal-foot caps
Slight swellings at the ends
ofbasal foot conical structures.
Multiciliated cell maturation
As motile cilia grow from each basal body, the cells are
polarized to generate a collective, efficient and uni-
directional fluid flow across the multiciliated epithe-
lium. The PCP requires integration of polarization
information at the subcellular, cellular and tissue levels,
which is mainly transduced to each cell through prefer-
ential binding of the four-pass transmembrane protein
complex VANGL–CELSR (Van Gogh–Flamingo in
Drosophila melano gaster) on one cell to the seven-pass
trans membrane WNT receptor complex frizzled (FZD)–
CELSR on its neighbouring cell. This is followed by the
selective recruitment of cytoplasmic PCP components
such as Prickle-like protein (PRICKLE), DVL and diversin
(alsoknown as ANKRD6), which further promote
asymmetric protein localization along the planaraxis115.
Establishment of initial polarization bias in multi-
ciliated tissues. In the X.laevis larvae epidermis and in
respiratory airways, oviduct and brain, PCP proteins are
already polarized before ciliogenesis80,96,97,98,116–118. Both
mechanical and chemical cues have been proposed to
determine the onset of planar organization (FIG.3a).
Forexample, gradients of WNT ligands are thought
to act as global cues for PCP119–121. In the mammal ian
brain, the early passive flow of CSF, based on pressure
differences that arise from the drainage of CSF through
theforamen of Monro, has been suggested to providethe
initial cue for the planar polarization of progenitor cells
by activating the transmembrane mechano sensory
receptors polycystin1 and poly cystin2 on the primary
cilia of radial glial cells122. However, knockout of these
proteins only partially disrupts PCP in these cells; thus,
other mechano receptors may participate inthe initial
polarization or other mechanisms may guide the axis
and direction of PCP across ciliated tissues. Interestingly,
it was recently shown in X.laevis that a mechanical force
exerted along the anterior– posterior axis can drive
global planar pattern ing of multi ciliated tissue123. It is
thus possible that a combin ation of mechanical forces
and chemical signals directs global planar patterning in
ciliated tissues.
Rotational polarity of motile cilia. The rot ationa l polar-
ity of motile cilia defines the orientation of planar ciliary
beating. As basal bodies dock at the apical membrane,
their orientation is already biased along the anterior-
to-posterior embryonic axis in X.laevis epidermis and
in the oral direction in the airway but is randomly orien-
ted in the mouse brain80,124. Basal body orientation is then
progressively refined to the correctorientationthrough
a mechanism coupling thePCP pathway and the hydro-
dynamic forces generated by the fluid flow itself80,98,124.
Interestingly, motile cilia in X.laevis epidermis are able to
change orientation even after refinement, whereas brain
ependymal cells are fixed after the refinement stage80,124.
The coiled-coil protein basal body orientation factor 1
(Bbof1) islocalized to the ciliary rootlet in X.laevis and
possibly involved inthe mainten ance of ciliary orien-
tation, although the corres ponding mechanism remains
unclear125. Cilia motility is required for axoneme orien-
tation in the X.laevis and mammal ian brain124,126,127,
butdoes not seem to be required in the airways127.
Cytoskeletal networks are involved in the rotational
polarity of motile cilia. The microtubules interact with
each basal body through their basal foot, which is
aspeci fic basal body appendage that is proposed to be a
microtubule organizing centre128,129. γ-tubulin, outer dense
fibre protein 2 (ODF2) and galectin3 are microtubule-
associated proteins that are localized on basal-foot caps.
They are associated with the minus end of micro tubules
at nucleation and anchoring sites on the basal body.
Interestingly, loss of galectin3 or ODF2 or treatment
with the microtubule-depolymerizing agent nocoda-
zole leads to failure of rotational polarization and basal
body alignment89,130–132. Moreover, nocodazole treatment
disrupts the asymmetric localization of PCP proteins
such as VANGL1, VANGL2, CELSR1 and PRICKLE2
Figure 3 | Different kinds of planar cell polarity in multiciliated tissues. a|Planar
polarization of multiciliated tissues is initiated by mechanical forces —passive fluid
flow sensed by the primary cilium or strain on tissues — that polarize primary cilia or
apical microtubules. The core planar cell polarity proteins (frizzled 3 (FZD3), FZD6,
cadherin EGF LAG seven-pass G-type receptors (CELSRs), vang-like proteins (VANGLs),
pyruvate kinase 1 (PK1; also known as PKLR) and PK2 (also known as PKM)) are then
distributed asymmetrically to the front or back of the cell with respect to the final
direction of ciliary beating. Motile cilia start beating in biased or random directions
(parts b and c). b|In Xenopus laevis epider mis and in mammalian airways a nd oviduc ts,
cilia cover the entire apical surface of multiciliated cells. Rotational polarity, which is
defined by the direction of the beating, is already biased along the anteroposterior
axisin X.laevis and in the oral direction in the airway, and is then refined by the
hydrodynamic forces generated by the ciliary beating itself and/or by the planar cell
polarity pathway. c|In brai n ependym al cells, b asal bodi es dock at the apica l plasma
membrane in random orientation. Rotational polarity is set by a coupling between the
hydrodynamic forces generated by the ciliary beating itself and the planar cell polarity
pathway. Cilia are clustered on half of the apical surface in the downstream direction of
cerebrospinal fluid (CSF) flow, which is termed translational polarity. DVL, dishevelled.
Mechanical forces:
tissue strain and/or
passive CSF ow
b Rotational polarity in airways and oviduct
a Initial global planar patterning
Centrosome
c Rotational and translational polarity in cerebral ventricles
Planar cell polarity
and uid ow
Planar cell polarity
andor uid ow
Microtubules FZD3, FZD6 CELSRs Actin, DVL1, DVL2
VANGLs, PK1, PK2
Basal
body
Nature Reviews | Molecular Cell Biology
Secretory cell
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Myosin Id
A short-tailed classI myosin; a
monomeric actin-based motor.
Metachronal
Self-organization of ciliary
beating in a wave-like pattern.
in epithelial cells97,116, suggesting that microtubule net-
works contribute to the asymmetric localization of
these proteins. Loss of centrin2, which is a core basal
bodyprotein, or sperm- associated antigen6 (SPAG6),
which is a core axoneme protein, leads to an abnormal
number of basal feet and defects in PCP, although the
mechanism remains to bedetermined133,134.
In multiciliated cells, actin networks are organized in
two pools that are found in the apical (centriolar) and
the subapical surface of the cell (just beneath centri oles).
Disruption of actin networks with low doses of cytocha-
lasin D or the absence of CELSR2 leads to disorganized
distributions of basal bodies, metachronal ciliary beating
defects and rotational polarity defects, as motile cilia fail
to undergo beating-direction refinement89,97. Similarly,
loss of myosinId, which is localized in the cortex actin net-
work, disrupts the asymmetric localization of VANGL1 in
the cerebral trachea and leads to rotational defects135. Two
cytosolic DVL proteins, DVL1 and DVL2, associate with
basal bodies in X.laevis epidermis, mammal ian trachea
and multi ciliated cells of the brain and are required for
the establishment and refinement of cili ary polariza-
tion36,80,92,97,117,136. Interestingly, ablation of the polarity
proteins VANGL2, FZD3, CELSR2 or CELSR3 affects
the orientation of cili ary beating, suggesting that these
proteins act upstream of rotational polarity establishment
associated with the basal bodies80,92,96,97,116,117(FIG.3b).
In contrast to X.laevis epidermis and mammalian
trachea and oviducts, in which multiciliated cells are
entirely covered by cilia, brain ependymal cilia are clus-
tered on half of the apical surface, in the downstream
direction of CSF flow, termed translational polarity65
(FIG.3c). This polarity is established at the precursor-cell
stage and involves primar y cilia signalling and the
transmembrane mechanoreceptors polycystin1 and
polycystin2 (REF.122). Rostral migration of basal bodies
in ependymal multiciliated cells requires an actin-based
motor protein complex called non-muscle myosin II136,
myosin Id135 and the PCP proteins VANGL2, FZD3,
CELSR2, CELSR3 and DVL97,117.
Control of ciliary beating. Assembly of 9 + 2 cilia self-
organizes their beating through hydrodynamic coupling
and the cytoskeleton, to form metachronal waves that
increase propulsion efficiency89,137,138. Inthe airways,
ciliary beat frequency can be modulated by numerous
inhaled chemicals139. For example, bitter compounds
increase the frequency of ciliary beating140. Mechanical
constraints are also involved. Increased fluid viscosity
induces entry of calcium into the cells and subsequent
activation of cilia in the oviducts, but decreases ciliary
beat amplitude in the brain and in the airways141–143.
Polycystin1 and polycystin2 are localized on motile
cilia in the oviducts, brain and airways, and might regu-
late cilia beat frequency by sensing external fluid flow
and intracellular calcium flux144,145. In addition, inflam-
mation mediators such as interleukins and hormones
(for example, progesterone) modulate ciliary beat fre-
quency in the airways and oviduct139,146–149. Inthe brain,
ATP d ecre ases c ilia ry b eat f requ enc y, wh ere as se ro-
tonin and melanin-concentrating hormone have the
oppositeeffect150,151.
Functions of multiciliated cells
Multiciliated cells fulfil crucial and diverse functions in
different organs. Defects in multiciliated cell develop-
ment or function lead to severe disorders such as devel-
opmental brain defects, irreversible lung failure and
subfertility (BOX1). Ciliary beating enables protective
mucus clearance in the airway, circulation of CSF in the
adult brain and ovum transport in the oviduct. Inaddi-
tion, less well studied and atypical multiciliated cells
might also regulate essential functions in other
tissues
and organs (BOXES2,3).
Box 1 | Genetic disorders of motile cilia
Primary cilia dyskinesia (PCD), previously called immotile-cilia syndrome or Kartagener
syndrome in people with left–right asymmetry defects219, is an autosomal recessive
disorder with an estimated prevalence of around 1 in 4,000 to 1 in 50,000 people220.
Almost 40 genes are known to cause cilium motility defects in individuals with PCD,
butthese explain only 50% of the cases221. The associated cellular defects range from
abnormalities of ciliary beating pattern or frequency to total immotility.
Individuals diagnosed with reduced generation of motile cilia (RGMC) exhibit a
totalabsence of cilia or a drastic decrease in cilia number owing to the absence or
mislocalization of ciliary basal bodies58,81–83,222–239. This condition, also called ciliary
aplasia/hypoplasia or acilia syndrome, is prevalent in 1–6% of individuals with
PCD82,222,233,235. Causal mutations were recently described in genes involved in centriole
generation58,81. PCD and RGMC share major clinical symptoms, which are described
below. Disease severity is highly variable, and the relationship between genotype,
ciliadefect and phenotype is unclear221.
Brain-associated defects. Hydrocephalus is characterized by the accumulation of
cerebrospinal fluid in the ventricular cavities and the mechanical compression
of brain parenchyma240. Numerous studies observed an association between ciliary
aplasia82,222,227,232 or dyski nesia241–248 and hydrocephalus. In two cohorts of individuals with
PCD, 2.5–3.5% of the individuals had hydrocephalus242,249. In smaller cohorts of RGMC,
theprevalence was 10–50%82,222,223,236. All of the studies indicate a higher prevalence of
hydrocephalus in PCD than in the general population, suggesting that cilium defects
increase morbidity. Further studies are needed to determine whether ciliary aplasia results
in a more severe phenotype than ciliary dyskinesia. Defects in motile cilia could also be
involved in the pathophysiology of Alzheimer disease and Huntington disease250,251,252,253,254.
Fertility. Although women with dyskinetic cilia can be fertile244,255,256, some studies
reporta correlation b etween cilia absence or dy skinesia and female steri lity257–262.
Studies in small cohorts report a higher prevalence of infertility in people with PCD
orRGMC81,82,249,263. Penetrance seems to be variable and may depend on the type of
mutation and physiological context. It is often stated that cilium defects can lead to
ectopic pregnancies but, to date, only two cases of ectopic pregnancy associated with
ciliary dyskinesia have been described262,264. Accurate assessment of fertility problems
ina large cohort of women with PCD or RGMC is needed.
A large proportion of men carrying a PCD mutation are sterile, but this is attributed
tosperm flagella dyskinesia. Fertility in people with RGMC mutations, which only affect
the number of cilia of multiciliated cells and not the sperm flagella, needs to be assessed.
Ofnote, a case of azoospermia in a male with ciliary aplasia and normal spermatozoa
hasbeen reported238.
Mucociliary clearance-associated defects. A universal feature of cilium defects is the
recurren ce of airway i nfections st arting from ear ly childhood as a result of in adequate
mucociliary clearance, mucal stasis and secondary pathogen contamination in the
Eustachian tubes, sinonasal tracts, throat and pulmonary tracts265. Recurrent infections
lead to mucus gland hypertrophy, further impairing mucociliary clearance266. Cilium
defects underlie chronic infections such as sinusitis, rhinitis and otitis media with
effusion. The most serious consequence is the occurrence of chronic lower respiratory
tract infections that can eventually lead to bronchiectasis — a permanent dilation of the
airway and thickening of the bronchial wall that is conductive to further infection. This
can, in the most severe cases, necessitate lung transplantation or lead to early death265.
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Glycocalyx
The polysaccharide matrix that
surrounds the cell membrane.
Sperm capacitation
Physiological changes of
spermatozoa that give them
the ability to penetrate
andfertilize an egg.
Ears, nose, throat and lungs. To protect against co ntinu-
ous exposure to the pathogens, particles and toxic chem-
icals in inhaled air, secretory cells covering the epithelium
of the ear–nose–throat and lung sphere continually pro-
duce liquids that function as physical barriers against
inhaled elements and secrete anti microbial agents that
act as an immune defence152–154. Inthe tracheo- bronchial
tree, secretory cells are interspersed with ciliated cells in
a ratio approaching 50:50, although the proportion of
cili ated cells decreases towards the distal end of the bron-
chioles155–157. Together with coughs, constantly beating
cilia provide the necessary force to transport pathogen-
filled airway liquids out of the respiratory system to the
pharynx, where they are swallowed152 (FIG.4). The nasal
cavity and para nasal sinuses, together with the tym-
panic cavity and the Eustachian tubes, are covered with
a comparable mucociliary epithelium158–161. Inaddition
to pressure differences during respiration and swallow-
ing and drainage via blood and lymph, acilium- driven
mucus blanket helps to keep the ear and nose sphere
sterile162–164 by sweeping inhaled particles towards the
pharynx (FIG.4).
The mucociliary system consists of two compo-
nents: awatery periciliary layer that provides a favour-
able environ ment for cilia beating and a gel-like mucus
that is composed of water and glycosylated proteins
called mucins. The mucus lies above the tip of motile
cilia, where it traps particles152,154 (FIG.4). Densely
packed macro molecules, tethered to respiratory cilia,
are thought to stabilize the two-layer system by form-
ing a mesh that prevents mucus and inhaled particles
from penetrating into the interciliary space165. In addi-
tion, intermolecular repulsion prevents the osmotically
active mucus from compressing the periciliary layer142.
Mucus hydration (viscosity) and ciliary beat frequency
are linked through Ca2+ and cAMP signalling, which
enable the continu ous adaptation of the two parameters
for optimal mucusclearance142,166.
In addition to mucus clearance, a recent study inzebra-
fish suggests that motile-cilium-mediated flow in the
nasal cavity improves the sensitivity and temporal resolu-
tion of the sensory cilia of olfactory neurons167. Whether
this applies in non-aquatic vertebrates, in which the olfac-
tory region is also located in a motile-cilium-covered
epithelium159, is unknown.
Reproductive tracts. Multiciliated cells are found from
the uterine cavity to the oviduct fimbria. Owing to men-
struation, the uterine epithelium reforms at each ovar-
ian cycle. The proportion of ciliated cells peaks at 20%
in both the uterine glands and lumen at the middle of
the ovarian cycle22,168–170. Although modest de-ciliation
has been described in some parts of the oviduct epi-
thelium during the secretory phase13,171, most ciliated
cells remain intact in the oviducts in the course of the
ovarian cycle13,172–175. However, the proportion of cili-
ated cells varies along the length of the oviduct, from
up to 80% of the cells in the fimbrium to 30% in the
isthmus13,172,175. The percentage of ciliated cells decreases
during pregnancy and with steroid contraception13,174.
The function of cilia in the uterus is unknown, but
they are thought to transport secreted material from
glandular and luminal secretory cells. In the oviduct,
ciliary beating is the principal driver of oocyte trans-
port towards the uterine cavity176. Cilia are proposed to
interact with the ovum through a ‘crown’ of glycocalyx
that is present on the tip of cilia177,178. Consistent with
the role of cilia in ovum transport, the beat frequency of
cilia increases after ovulation175,179, a process that is pro-
posed to be triggered by follicular fluid180. Intercourse
up to 6days before ovulation can lead to pregnancies in
humans181, so sperm can be stored in the female repro-
ductive tract, in particular by multiciliated cells in the
oviduct. Long-term sperm storage is used by various
animals, including mammals, in which copulation and
ovulation are asynchro nous182. Although controversial183,
numerous studies found a close interaction between
sperm heads and cilia in human oviducts both invitro
and invivo184–186. Inaddition, direct contact between
oviduct and sperm cells is thought to increase sperm
survival and to be involved in sperm capacitation, at least
invitro187–189. In cows and pigs, capacitated sperm cells
are less prompt to attach to multiciliated cells in the
oviduct 189,190. Tubal epithelium may thus prolong the
availability of viable sperm and, by inducing capacita-
tion, allow their release for successful fertilization. The
recent development of human fallopian tube organoids
will help to test these hypotheses23.
In the male reproductive tract, efferent ducts trans-
port fluid and spermatozoa from the testis, where they
are produced, to the epididymis. The lumen is lined by a
single epithelium composed of an increasing proportion
— from the testis to the epididymis — of multiciliated
cells191–193. The main function of efferent duct epithelium
Box 2 | Under-studied multiciliated cells
Several tissues have multiciliated epithelia of unknown function in physiological or
pathological conditions.
Kidney. Although multiciliated cells are not uncommon in the renal tubules of simpler
forms of animals, they are not usually found in the renal tubules of humans. Numerous
studies, however, have detected multiciliated cells that are typical of ciliated epithelia
in the adult human kidney267–272 and urethra 273–275 in a numbe r of pathol ogies.
Thissuggests that human kidney cells have retained the capacity for multiciliation.
Thiscapacity might be exploited in pathological conditions to facilitate filtration,
asinlower species. Interestingly, the efferent duct, which possesses a ciliated
epithelium that resorbs testis fluid, originates from the embryonic kidney194.
Oesophagus. Multiciliated cells line the human fetal oesophagus. Their proportion
increases gradually and peaks at around the sixteenth gestational week, then decreases
to practically zero at birth, the time of first feeding276,277, potentially by desquamation278.
A similar transitory developmental process exists in fish, birds, amphibians and
rodents279,280. Beating cilia may drive the circulation of embryonic fluids and possibly
participate in oesophageal development228. Oesophageal and tracheal cells both
differentiate from p63‑expressing progenitors in the foregut endoderm281. As adult
p63‑knockout mice retain a ciliated oesophageal epithelium, p63 expression might
regulat e the transito ry ciliation of the fetal oeso phagus25.
Spinal canal. Multiciliated cells are present in the zebrafish, rabbit, macaque and
human spinal cord282–285 but are absent in rodents. They were proposed to be involved in
propelling cerebrospinal fluid. A recent study in zebrafish revealed that cilium-motility
mutants develop the spinal deformity idiopathic scoliosis283, and scoliosis was also
reporte d in primary ci liary dyskin esia228,286–288. Whether cilium-driven cerebrospinal fluid
flow in the spine is involved in skeletal development needs to be investigated.
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Interstitial fluids
Extracellular fluids formed
from plasma at the capillary
walls in th e brain.
Near-wall CSF circulation
Movement of cerebrospinal
fluid (CSF) close to the
ventricular walls that is
opposed to the movement
ofCSF in the central region of
the cavity.
is to stir and resorb testis fluid, thereby concentrating
the sperm191,194,195. Fluid and sperm are transported
towards the epididymis along a gradient of pressure
that is generated by seminiferous secretions, contrac-
tions and fluid absorption by the efferent ducts196–198.
Instead of transporting gametes, cilia are thought to cre-
ate a reflux196 and have been reported to beat in opposite
directions depending on the side of the lumen on which
they are located199. Mathematical modelling predicted
that this reflux would be bypassed when the viscosity
of sperm increases as a result of its concentration196,
thereby allowing only sufficiently concentrated sperm
to reach the epididymis. Additional measurements of
cilia motility in the efferent duct are needed to confirm
this hypothesis.
The nervous system. In the human adult central nervous
system, half a litre of CSF is secreted every day into the
brain ventricular system by the epithelial cells of the cho-
roid plexus (BOX3). In addition to its role as a cushion,
CSF distributes secreted trophic and metabolic signals
throughout the ventricular system and clears the brain
of toxins and waste200. Multiciliated ependymal cells
cover the lumen of most of the ventricular network and
were traditionally thought to drive CSF propulsion201,202.
However, the contribution of ciliary beating to bulk fluid
propulsion in the brain ventricles might, in fact, depend
on the species and, in particular, on the size of the brain
cavities. Indeed, in small mammals such as rodents,
ependymal ciliary beating seems to be crucial for CSF
propulsion, as evidenced by the systematic develop-
ment of hydrocephalus in cilium-defective mutant mice.
However, recent evidence in humans suggests that the
main elements driving bulk CSF circulation are: first,
the pressure gradient created by secretion of the fluid
at the back of the ventricles and outflow at the front;
second, bidirectional fluid exchanges with interstitial
fluids; andthird, changes in arterial pressure during the
heartbeat cycle203,204. Confirming that bulk flow is not
driven by cilia, flow rates, measured by phase contrast
magnetic resonance imaging (MRI) in humans, show
that movement of the CSF is pulsatile and correlated
with heartbeat; under some circumstances, net flow
can even be reversed with flow into rather than outside
thebrain203,204.
In humans, cilia are thus more likely to be important
for near-wall CSF circulation. In support of this hypothesis,
computational simulations of fluid dynamics in humans
using subject-specific parameters derived from MRI pre-
dicted that microscale near-wall flow is dominated by
the action of ependymal cilia. By contrast, macroscale
CSF dynamics in the central regions of the ventricles
are predicted to be the consequence of wall motion and
choroid plexus pulsations205. Near-wall flow circula-
tion was observed long ago in humans202 and recently
described in more detail in mouse, rat and pig samples206.
These studies show that multiciliated cells are arranged
in modules and beat in different, sometimes contrary
directions, thus creating separate flow compartments.
Computational fluid modelling based on the geometry
of the human ventricles and their connecting aque-
ducts extracted from MRI scans predicted that such
recircu lation regions exist as a result of macroscale fluid
dynamics207–209. Because cilium orientation in the brain
is determined during development in response to hydro-
dynamic forces80, macro scale fluid dynamics could set
up the orientation of cilia in separated modules.
The complex flows may result in differential distrib-
ution of signalling molecules in the ventricles that
could account for preferential routes of communication
between different brain regions206,209, but this remains
to be tested experimentally. For example, transport
of the chemo-repulsive SLIT proteins by the CSF has
Box 3 | Atypical multiciliated cells
In the nervous system, multiciliated cells with an atypical cilium structure or number
exist. These include choroid plexus multiciliated cells, biciliated cells in the brain
andthe spinal cord canal, and multiciliated olfactory neurons.
Choroid plexus multiciliated cells. The choroid plexus is a highly vascularized secretory
epithelium found in the lateral, third and fourth brain ventricles in continuity with the
ependyma. Choroid plexus epithelial cells specialize in the production of cerebrospinal
fluid (CSF)200. Each cell harbours a tuft of less than 20 cilia, which are thought to be
involved in the regulation of ion transport and CSF production289,290. The differentiation
of choroid plexus cells seems to involve the Notch, multicilin and forkhead box proteinJ1
(FOXJ1) signalling pathway, which is typically involved in the formation of multiple
motile cilia291. However, the identity of choroidal cilia is controversial. Although the first
electron microscopy studies detected a 9 + 2 axoneme in humans and other species292–294,
convincing images have since accumulated showing a 9 + 0 axonemal structure289,295–297.
The authors of the earliest studies might have been misled by the organization of
microtubule doublets in choroid plexus epithelial cells, where doublets are positioned
towards the centre of the axoneme295. Alternatively, putative differences between
species and/or the existence of a shift from a 9 + 2 motile pattern to the immotile 9 + 0
pattern during the perinatal period might account for these discrepancies298,299;
ifconfirmed, this transition in axoneme organization would be unique.
Biciliated cells in the brain and the spinal cord canal. Unusual biciliated ependymal
cellshave been observed in brain ventricles and in the central canal of the spinal cord.
In the mouse brain, they are rare in the lateral ventricle but form a distinct biciliated
epithelium extending along the ventral third into the fourth ventricle214,300. Their
function is as yet undetermined. In the central canal of the rodent spinal cord, most
ependymal cells harbour two 9 + 2 cilia (a small proportion of cells have one, three or
four 9 + 2 cilia) that project into the spinal canal. In rabbits, macaques and humans, uni-
and biciliated cells are also observed, but most spinal ependymal cells are multiciliated
(15–30 cilia)282,284,285. Biciliated cells in macaques and rodents divide and are thought to
contribute to extending the length of the central canal during growth282,301 and to the
formation of scar-forming glia and oligodendrocytes when the spinal cord is injured302.
Dividing biciliated cells have not been observed in humans282. Biciliated cells can also
help to propel the CSF and/or act as sensory organelles that monitor its composition301.
Multiciliated olfactory neurons. The olfactory epithelium is a small area located in
therespiratory epithelium in the upper region of the nasal cavities. Olfactory neurons
contact the nasal cavity through their dendritic knobs and synapses with secondary
neurons in the olfactory bulb. Each neuron projects multiple (10–30) sensory cilia, which
are essential for the conversion of external chemical stimuli into intracellular electrical
responses303. Olfactory cilia form a dense, intermingled meshwork covering the
epithelium159. Their length (1–110 mm (REFS303,304)) is correlated with their sensitivity
to odour in rodents305. These cilia contain a unique axonemal structure. Whereas their
proximal segment has a 9 + 2 axoneme and the typical cilium diameter of 0.3 mm, cilia
taper down to a diameter of0.1 mm as they extend over the epithelial surface. Their
axonemal structure thus shifts gradually to nine and then to two singlets159,306.
Mammalian olfactory cilia lack dynein arms and are therefore immotile303. Despite the
essential function of olfactory cilia307–311, the molecular mechanism of their formation is
undetermined. The absence of deuterosomes and the presence of centriolar rosettes
observed by electron microscopy on differentiating neurons suggest that centrioles are
formed through a specific centriolar pathway312–314.
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ǟɥƐƎƏƗɥ!,(++-ɥ4 +(2'#12ɥ(,(3#"Ʀɥ/13ɥ.$ɥ/1(-%#1ɥ341#ƥɥ++ɥ1(%'32ɥ1#2#15#"ƥ
Basal process
Cell extension from the cell
body that contacts the pial
surface of the brain.
been shown, in the adult mouse, to create a gradient
in the neuro genic region lying beneath the ependy-
mal epithelium. This gradient has been proposed to be
responsible for the migration of new neurons towards
the olfactorybulb210,211.
The heartbeat, breathing and state of awareness
modify the complex flows206,208,212. Given the major
contrib ution of molecules in the CSF to brain develop-
ment and homeostasis200,213, one can hypothesize that
cilium-driven near-wall CSF circulation represents an
underestimated cell-extrinsic route of regulating brain
function and neurogenesis. In adult rodents, neural
stem cells contact the ventricle by extending a thin basal
process with a single, non-motile sensory cilium at the
centre of rosettes of ependymal cells214. Ependymal
cells secrete factors that modulate adult neurogenesis,
such as noggin215, the chemokine stromal cell-derived
factor1 (SDF1)216 and pigment epithelium-derived fac-
tor (PEDF)217,218. Together, these observations suggest the
existence of direct biochemical and/or physical regulation
of adult neurogenesis by ependymalcells.
Conclusions and open questions
Multici liated cells form a prote ctive s heath over the cav-
ities of a number of organs and also help to maintain
their homeostasis and proper functioning. Depending
on the organ, these cells may undergo rapid turnover
or last for years. The fine mechanisms of their develop-
ment and maintenance at both the cell and tissue levels
are beginning to be understood. However, a number of
questions still need to be answered to fully understand
the molecular mechanisms regulating the formation of
centrioles, the alignment of ciliary beating at cell and
tissue levels, and the renewal or maintenance of these
cells throughout life. More specifically, how are the
major regulators of centriole duplication in cycling cells
repurposed to permit the amplification of centrioles?
How do multiciliated cells orient and coordinate their
ciliary beating at the tissue level to produce efficient fluid
flow in the cavities of each organ? How are these cells
replaced throughout life? Recent advances in live imag-
ing and the development of cultured organoids should
enable us to address these fascinatingquestions.
Figure 4 | Locations and proposed functions of multiciliated cells in the human body. In the mucociliary system
(topright), the convergence of cilium-powered mucus transport (arrows) from the middle ear, the bronchial tree and the
paranasal sinuses towards the pharynx helps to keep the entire ear–nose–throat–bronchial sphere free from airborne
hazards. The mucociliary system consists of a two-component ‘gel-on-a-brush’ system: a watery periciliary layer of motile
cilia and a gel-like mucus, which is composed of water and glycosylated proteins called mucins and lies above the tip of
motile cilia. Densely packed macromolecules that are tethered to respiratory cilia are thought to stabilize the two-layer
system by forming a mesh that prevents mucus and inhaled particles from penetrating into the interciliary space.
In the female reproductive tract (bottom right), ovum pick-up by ciliated cells is needed for efficient transport towards the
uterine cavity. In human efferent ducts (bottom left), multiciliated cells resorb testis fluid, and cilia are thought to create
areflux that allows only sufficiently concentrated sperm to pass. Whereas the function of multiciliated cells in the
spinalcanal is unknown (top left), in the lateral, third and fourth brain ventricles, ependymal cilia contribute to near-wall
cerebrospinal fluid (CSF) propulsion. The functions of multiciliated cells in the fetal oesophagus and in pathological
kidneyconditions areunknown.
Brain Middle ear
Eustachian tubes
Nasal cavities
Paranasal sinuses
Trachea
Lung
Oviduct
Ampulla Isthmus
Fimbria
Ovary
Mucus clearance
Ovum transport
Naso-pharynx
Pharynx = swallowed
Larynx
Gel-on-a-brush
Mucus
layer
Periciliary
layer
Spinal canal
Fetal oesophagus
Pathological
kidney conditions
Male eerent ducts
Concentrating the sperm
y uid reasorption
Unknown function
CSF near-wall circulation
Lateral
Uterus
Third
Fourth
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Ackno wle dgem ent s
Research in the Spassky laboratory was financed by the
Institut National pour la Santé Et la Recherche Médicale
(INSERM), the Centre National de la Recherche Scientifique
(CNRS), the Ecole Normale Supérieure (ENS), the Agence
Nationale de la Recherche (ANR-12-BSV4-0006andANRJC
JC-15-CE13-0005-01), the European Research Council (ERC
Consolidator grant 647466), the Fondation pour la
Recherche Médicale (FRM20140329547), the Cancéropôle
Ile-de-France (2014-1-PL BIO-11-INSERM 12–1) and the
Fondation Pierre-Gilles de Gennes (FPGG03). The authors are
grateful to the members of their laboratory for insightful and
stimulating discussions.
Competing interests statement
The authors declare no competing interests.
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