Developmental Cell, Vol. 7, 401–412, September, 2004, Copyright 2004 by Cell Press
Directional Cell Migration Establishes
the Axes of Planar Polarity
in the Posterior Lateral-Line Organ of the Zebrafish
The first mitosis, which is invariably oriented parallel to
the anteroposterior axis of the fly, is controlled by Friz-
zled and other components of the planar polarity signal-
ing pathway (Gho and Schweisguth, 1998).
Although the proteins involved in interpretation of the
polarizing signals have been widely studied, the cell-
biological mechanisms that lead to planar polarization
are not well understood (Keller, 2002). The mechanore-
ceptive hair cell of the vertebrate internal ear provides
ment in amenable to investigation (Lewis and Davies,
2002). A hair cell takes its name from the hundred or so
cylindrical processes constituting the hair bundle that
projects from the cellular apex (for a review see Huds-
peth, 1989). A single true cilium, the kinocilium, occurs
at the tall edge of the bundle. The remaining processes,
the actin-rich stereocilia, are organized in order of in-
creasing height across the apical surface of each cell,
so that the hair bundle’s top surface is bevelled like a
hypodermic needle. The axis of morphological polarity
corresponds to the cell’s axis of excitability (Shotwell
et al., 1981). A positive deflection, which moves the
bundle toward its tall edge, opens transduction chan-
nels, allows cations to flow into the cell, and thereby
depolarizes the plasma membrane. Deflection in the op-
posite direction leads to hyperpolarization. The senses
of hearing and equilibrium thus rely on the exquisite
precision with which tens to thousands of hair cells are
oriented relative to one another across the sensory epi-
Some aquatic vertebrates sense directional water
closely related to that of the ear (Montgomery et al.,
1997). This organ comprises a stereotyped array of sen-
sory clusters called neuromasts (Metcalfe et al., 1985;
Montgomery et al., 1997). Haircells and supporting cells
form the core of each neuromast, which is innervated
by sensory neurons projecting to the central nervous
system (Metcalfe, 1985; Alexandre and Ghysen, 1999).
In the zebrafish, there are two main components, the
anterior andposterior lateral-line organs,which develop
from cephalic epithelial thickenings called neurogenic
ectodermal cells (Gompel et al., 2001). A group of 30
cells forms the precursors of a ganglion and of one
neuromast (L1), whereas the remainder of the cells con-
stitute a first primordium that initiates a posteriorward
migration around 20 hr postfertilization (hpf) (Sapede et
al., 2002). Cells of this primordium express the CXCR-
4b chemokine receptor, which allows them to follow the
horizontal myoseptum along a path formed by the Sdf-
1a chemokine (David et al., 2002; Li et al., 2004). By 40
hpf, the first primordium has completed its journey to
the tail of the fish, leaving behind seven to nine pro-
neuromasts. Soon thereafter, a second, smaller primor-
dium begins to migrate along the same path, depositing
a few pro-neuromasts over the rostral part of the trunk.
The entire complement of neuromasts later migrates
ventrally to reach its final location several neuromast
Herna ´n Lo ´pez-Schier, Catherine J. Starr,
James A. Kappler, Richard Kollmar,1
and A.J. Hudspeth*
Laboratory of Sensory Neuroscience and
Howard Hughes Medical Institute
The Rockefeller University
1230 York Avenue
New York, New York 10021
The proper orientation of mechanosensory hair cells
along the lateral-line organ of a fish or amphibian is
essential for the animal’s ability to sense directional
water movements. Within the sensory epithelium, hair
cells are polarized in a stereotyped manner, but the
mechanisms that control their alignment relative to
the body axes are unknown. We have found, however,
that neuromasts can be oriented either parallel or per-
pendicular to the anteroposterior body axis. By char-
acterizing the strauss mutant zebrafish line and by
romasts of these two orientations originate from, re-
spectively, the first and second primordia. Further-
more, altering the migratory pathway of a primordium
reorients a neuromast’s axis of planar polarity. We
to the body axes is established through an interaction
between directional movementby primordial cells and
the timing of neuromast maturation.
The coordinated orientation of polarized cells within the
plane of an epithelium is termed planar cell polarity. The
stepsproceeding fromglobalcellorientation tointracel-
lular and cytoskeletal rearrangements along the axis of
polarization. Extensive genetic analyses in Drosophila
have identified several proteins involved in the control
elled form part of the core of the signaling pathway
(Strutt, 2003). These proteins integrate signals leading
to the alignment of hairs and bristles in the wing, notum,
and abdomen, as well as the orientation of ommatidia
in the eye (for reviews see Adler, 2002; Tree et al., 2002;
Uemura and Shimada, 2003). The global orientation of
cellular asymmetry relative to the main body axes is
exemplified by the cell divisions of the sensory-organ
precursors inthe Drosophila pupalnotum. Thefour cells
that form each sensory organ originate through two
rounds of mitosis from a single precursor cell called pI.
1Present address: Department of Molecular and Integrative Physiol-
ogy, University of Illinois at Urbana-Champaign, Urbana, Illinois
diameters below the horizontal myoseptum (Ledent,
2002; Sapede et al., 2002). The developing posterior
lateral-line organ therefore undergoes a net posterior
and ventral movement. By the fifth day of development,
all neuromasts contain functional hair cells, but those
generated by the second primordium can still be identi-
fied by their location and smaller size.
Numerous studies have described the developmental
and molecular basis of the formation and innervation of
the lateral-line organ in wild-type zebrafish and other
species. Few, however, have focused attention on the
development of planar polarity by hair cells in neuro-
masts (Rouse and Pickles, 1991). In particular, nothing
is known about the cell-biological basis of hair cells’
global orientation along an animal’s body. We report
here the initial results of our investigation of this issue,
which indicate that the trajectory of the primordial cells
bine to establish this orientation.
with a fluorescent compound, 4-Di-2-ASP, that enters
transducing hair cells (Figures 2A–2D). Using rhoda-
mine-phalloidin to mark filamentous actin in stereocilia
(Figures 1G and 2E) and an antibody against acetylated
tubulin to label each hair cell’s soma and kinocilium
(Figures1Gand 2F)(HaddonandLewis, 1996),wefound
that every neuromast possesses an axis along which all
the constituent hair cells align in parallel but opposing
orientations (Figures 2F and 2G) (Lewis and Davies,
2002). The orientation of this axis is highly conserved in
each specific neuromast. For more than 100 wild-type
fish analyzed at 6 dpf, hair bundles in neuromast L1 and
in L5–L9 were always aligned with the anteroposterior
axis of the body; we designate neuromasts of this con-
figuration “parallel.” By contrast, the hair bundles in
neuromasts L2, L3, and often L4 oriented their hair bun-
dles orthogonally to this axis (Figure 2H); we term such
neuromasts “perpendicular.” In about 20% of the ani-
mals analyzed, however, hair bundles in L4 were polar-
ized parallel to the anteroposterior axis.
rior lateral-line organ displays three levels of planar cell
polarity. The first level, defined by the eccentric location
of the kinocilium in the hair bundle, corresponds to the
vectorial mechanosensitivity of the individual hair cell
reflects the axis of hair-cell orientation within a neuro-
mast, that is, the orientation of individual hair cells with
respect to one another (Flock and Wersa ¨ll, 1962; Lewis
and Davies, 2002). The final level, which characterizes
each neuromast along the anteroposterior axis of the
animal, specifies the orientation of each neuromast with
respect to the animal’s main body axes. The highly con-
served organization of planar polarity along the sensory
organ implies that its establishment must be precisely
Morphogenesis of the Posterior Lateral-Line Organ
The first primordium of the posterior lateral-line organ
begins to migrate along the horizontal myoseptum at
20 hpf, depositing discrete groups of cells, the pro-
neuromasts, in a stereotyped pattern along the animal’s
body to its tail (Ledent, 2002; Sapede et al., 2002). Sen-
sory axons of the octavolateralis system closely follow
this moving primordium, pathfinding for glial cells of
neural-crest origin (Gilmour et al., 2002).
We have investigated the development of the lateral-
line organ with an antibody against the tight-junctional
component claudin b, whose transcript is highly ex-
et al., 2001). The antibody marks all the cells of the
migrating primordium, the pro-neuromasts, and a trail
of cells connecting them (Figures 1A–1C). To confirm
that these trailing cells derive from the primordium, we
injected single-cell embryos with caged fluorescein-
dextran and used ultraviolet-light irradiation to uncage
the fluorophore in primordial cells at the onset of migra-
tion. The following day, animals were fixed and the clau-
din b protein was immunolabeled. All green-fluorescent
trailingcells werealsointensely labeledby theantibody,
confirming their primordial origin (Figure 1D).
Using claudin b as a marker, we also confirmed the
observation that the sensory nerve fibers trace the tra-
jectory of the primordium (Gilmour et al., 2002, 2004).
The claudin b-positive cells lying between pro-neuro-
masts frequently contact the axons and sometimes en-
wrap them in a manner suggestive of Schwann cells
(Figures 1Eand 1F).They maytherefore representa glial
subtype of placodal origin that ensheathes the accom-
panying axons during migration (David et al., 2002). At
4 days postfertilization (dpf), claudin b becomes re-
marker for these cells identified in the zebrafish.
Neuromast Innervation Is Dispensable for Planar
Polarization of Hair Cells
volateralis system confers neuromast identity along the
anteroposterior axis, and thus sets the orientation of
different neuromasts. To do this, we used an antisense
morpholino directed against neurogenin-1 transcripts.
Neurogenin-1 is necessary for the differentiation of all
cranial ganglia, including the octavolateralis ganglion,
but not for the formation of neuromasts and hair cells
(Andermann, et al., 2002). Although neurogenin-1 mor-
phant larvae lacked the acetylated tubulin-positive ax-
ons that normally lie beneath neuromasts, planar polar-
ization by hair cells was unaffected (data not shown).
Thus, innervation appears dispensable for neuromast
strauss Mutant Fish Lack Perpendicular Neuromasts
To investigate the requirements for planar polarity in
the posterior lateral-line organ, we sought mutations
affecting hair-cell orientation in mutant strains identified
during a mutagenic screen. We identified one line,
strauss, that has no defects in the first two levels of
ularly oriented neuromasts. strauss mutants also de-
velop fewer neuromasts and have smaller eyes than
wild-type fish (Figures 3A–3D).
Development of Planar Polarity in Hair Cells
Functional hair cells in the posterior lateral-line organ
begin to appear in an anterior-to-posterior progression
starting at 48–52 hpf, as assessed by labeling animals
Cell Migration and Planar Polarity
Figure 1. Claudin b Labeling of Primordial Cells and Mature Supporting Cells in the Posterior Lateral-Line Organ
(A) In a confocal section at 28 hpf, antiserum against claudin b (Cldn b) strongly labels all cells of the first primordium. The trail of cells
between the primordium and the most recently deposited pro-neuromast is also marked.
(B) The leading neurites, labeled for acetylated tubulin (Ac tub), invade the first primordium at an early stage of migration.
(C) In a confocal section of neuromasts at 48 hpf, axons of the octavolateralis nerve extend along the trail of claudin b-positive cells.
(D) Labeling of the first primordium with uncaged fluorescein-dextran (Fl-dex) marks neuromasts and trailing cells, which are also positive for
(E and F) Claudin b-positive cells enwrap the extending sensory axons adjacent to a neuromast (E) and along the developing octavolateralis
nerve (F). Note several points of apparent contact between the claudin b-positive cells and the axons (arrowheads).
(G) Actin-rich hair bundles are evident at hair-cell apices (asterisk) in a lateral view of a 5 dpf neuromast. Anti-claudin b specifically labels
mature supporting cells in all neuromasts, whereas acetylated tubulin marks the hair-cell somata and kinocilia (not evident in this image) as
well as neurons delaminating from the basal epithelial surface (arrowhead).
(H) In an intensity profile of a top view of a neuromast, peripheral supporting cells accumulate high levels of claudin b, which is excluded
from the center of the organ where mature hair cells label for acetylated tubulin and rhodamine-phalloidin. The intensity scale is linear but
its units are arbitrary. In these and all subsequent illustrations, the animal’s anterior is oriented to the left and its dorsum is situated to the top.
Scale bars: 50 ?m in (A)–(D), 10 ?m in (E) and (F).
The initial development of the posterior lateral-line
organ is unaffected by the strauss mutation. Wild-type
and mutantanimals formfirst primordiaof identicalsize,
which migrate along the horizontal myoseptum at the
same time and speed (data not shown). There is no
difference in the number of neuromasts deposited by
fish, the first functional hair cells appear in pairs aligned
parallel to the animal’s anteroposterior axis (Figures 3E
and 3F). The axis of hair-bundle polarity is also consis-
tently aligned with this body axis (Figures 3G and 3H);
there are no perpendicular neuromasts at this stage.
During the fourth day of development, wild-type-fish
begin to form hair cells in two or three additional neuro-
masts near the rostral end of the organ (Figure 3C).
roposterior body axis (Figure 3I). In strauss mutant lar-
vae, by contrast, additional neuromasts rarely appear
Distinct Origins of Parallel and Perpendicular
Our observation that parallel and perpendicular neuro-
masts develop sequentially suggests that the latter ma-
ture more slowly or that they derive from different pri-
mordia. To distinguish between these possibilities, we
Figure 2. Hair-Cell Development and Polarity in the Posterior Lateral-Line Organ of Wild-Type Larvae
(A) When treated with the fluorescent compound 4-Di-2-ASP at 48–52 hpf, only one neuromast on the head (left arrowhead) and one on the
trunk (right arrowhead) display functional hair cells.
(B) By 60 hpf, mature hair cells appear in additional neuromasts in an anterior-to-posterior progression.
(C and D) In lateral (C) and ventral views (D) of a 6 dpf larva, all the neuromasts contain several functional hair cells. The L1 neuromasts are
out of the plane of focus in (D). Note the difference in the positions of equivalent neuromasts on the two sides of the transparent larva.
(E) A surface view of a preparation labeled with rhodamine-phalloidin shows hair bundles protruding from the centers of two neuromasts.
(F) The axis of hair-bundle polarity in neuromast L1 is demonstrated by labeling with rhodamine-phalloidin, which marks the actin in the
stereocilia, and with antiacetylated tubulin, which marks the eccentric kinocilia.
(G) Labeling with rhodamine-phalloidin alone demonstrates parallel cellular polarity by revealing a notch in each actin-rich cuticular plate
corresponding to the position of the kinocilium.
(H) Similar labeling reveals the perpendicular orientation of hair bundles in neuromast L2.
Scale bars: 100 ?m in (A)–(D), 10 ?m in (E)–(G).
used laser-mediated cell ablation to eliminate first-pri-
mordial cells at the onset of migration. As assessed by
labeling at 60 hpf with 4-Di-2-ASP, unilaterally treated
two or three neuromasts situated between somites 6
were always perpendicular. The control side of the fish
displayed a normal organization with both neuromast
polarities. In a converse experiment, elimination of the
out perpendicular neuromasts on their treated sides
(data not shown).
These results suggest that all parallel neuromasts
originate from the first primordium, whereas perpendic-
ular neuromasts stem from the second. To confirm this
Cell Migration and Planar Polarity
Figure 3. Absence of Perpendicular Neuromasts in strauss Mutant Larvae
(A and B) Differential-interference-contrast images of a wild-type larva (A) and a homozygous strauss larva (B) at 4 dpf demonstrate the small
eyes of the mutant.
(C and D) Treatment of the same animals with 4-Di-2-ASP reveals the presence of functional hair cells in mature neuromasts. Wild-type fish
form additional neuromasts (dashed circle) in the rostral part of the organ, whereas strauss mutant animals do not.
(E) At 48–60 hpf, the hair cells of neuromasts appear in pairs (arrowheads).
(F and G) When labeled with 4-Di-2-ASP (F) or with an anti-parvalbumin 3 antiserum and rhodamine-phalloidin (G), these early neuromasts
are seen to be aligned parallel to the anteroposterior body axis.
(H) A higher-magnification view of a neuromast shows the opposing orientations of the two hair bundles whose axis of planar polarity runs
parallel with the axis of hair-cell development.
(I) A high-magnification view of the neuromast circled in (C) reveals the perpendicular development of the first two hair cells.
Scale bars: 100 ?m in (A)–(E), 1 ?m in (F)–(I).
inference, we marked first-primordial cells by uncaging
fluorescein-dextran in them at 20–24 hpf. At 5 dpf, we
consistently found that green-fluorescent cells popu-
lated only parallel neuromasts and the trailing cells con-
necting them, whereas perpendicular neuromasts were
pendicular neuromasts originate exclusively from the
second primordium. Variability in the number of pro-
neuromasts deposited by the second primordium ex-
plains why we observe two (L2–L3) or sometimes three
Figure 4. Origin of Parallel and Perpendicular Neuromasts
(A) After ablation of the first primordium on one side of a wild-type larva, treatment with the fluorescent compound 4-Di-2-ASP at 60 hpf
labels neuromasts on only the untreated side (compare Figure 2B).
(B) At 6 dpf, the treated side develops a single mature neuromast in the rostral part of the posterior lateral-line organ (arrowhead).
(C) Uncaging of fluorescein-dextran in the first primordium labels a neuromast (L1) and the trail of cells left by that primordium (green), whereas
a neuromast derived from the second primordium (L2) is not labeled.
(D and E) Actin labeling of the neuromasts depicted in (C) shows the parallel orientation of hair bundles in the fluorescein-positive neuromast
L1 (D) and the perpendicular orientation of those in fluorescein-negative neuromast L2 (E).
Scale bars: 10 ?m in (C)–(E).