Volume 7, Issue 3, Pages 401-412 (September 2004) Directional Cell Migration Establishes the Axes of Planar Polarity in the Posterior Lateral-Line Organ of the Zebrafish  Hernán López-Schier, Catherine J. Starr, James A. Kappler, Richard Kollmar, A.J. Hudspeth  Developmental Cell  Volume 7, Issue 3, Pages 401-412 (September 2004) DOI: 10.1016/j.devcel.2004.07.018

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 claudin b. (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 μm in (A)–(D), 10 μm in (E) and (F). Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)

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 μm in (A)–(D), 10 μm in (E)–(G). Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)

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 μm in (A)–(E), 1 μm in (F)–(I). Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)

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 μm in (C)–(E). Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)

Figure 5 Migration of Pro-Neuromasts Derived from the Second Primordium in Wild-Type and strauss Larvae (A) Labeling for claudin b marks the rostral end of the posterior lateral-line organ in a wild-type fish at 56 hpf. The arrow marks the initial location of the second primordium a few cell diameters posterior and dorsal to that of the first. The initial neuromast derived from the second primordium migrates ventrally and depresses the trail of claudin b-positive cells connecting adjacent first-primordial neuromasts (arrowhead). (B) Labeling for parvalbumin 3 demonstrates mature hair cells only in neuromasts derived from the first primordium (L1 and L3), whereas claudin b immunoreactivity additionally marks the second primordium and a pro-neuromast (L2) derived from it. (C) In a detailed view of the initial neuromast deposited by the second primordium, the arrowheads indicate the trail of claudin b-positive cells left by the first primordium. (D) Prior to its ventral migration, a neuromast deposited at the trailing edge of the second primordium already contacts the trail of claudin b-positive cells derived from the first primordium (arrowheads). (E and F) Of the five rostral neuromasts in a wild-type fish (dashed circles in [F]), two (L2 and L3) lie about two neuromast diameters below the horizontal myoseptum. (G and H) strauss mutants lack these ventrally located neuromasts; instead, all neuromasts (dashed circles in [H]) remain located close to the horizontal myoseptum. Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)

Figure 6 Reorientation of Neuromasts Associated with the Trajectory of Primordial Cells (A) Neuromasts assume random positions in a slow muscle omitted mutant larva labeled with 4-Di-2-ASP. (B) A mutant fish shows the atypical migration path followed by the primordium, which had initially migrated toward the posterior, then turned back anteriorly. In the inset, labeling of actin with rhodamine-phalloidin shows that the first two levels of planar cell polarity develop normally. (C) A fused somites mutant fish, labeled for acetylated tubulin (green) and actin (red), shows wayward migration of the posterior lateral-line organ (arrow) with a single neuromast at the end (arrowhead). (D) In a high-magnification view of the same neuromast, axons labeled for acetylated tubulin (green) reveal the trajectory of the primordium. (E) A higher-magnification view of the neuromast in (C) reveals hair cells that are polarized perpendicular to the anteroposterior body axis but are oriented along the trajectory of the axons. (F and G) Comparison of a wild-type larva (F) with a parachute/Ncad mutant larva (G) at 4 dpf demonstates that the mutant fish is shorter, misshapen, and displays defects in its trunk musculature. (H) Actin labeling reveals that the planar polarization of neuromasts in the mutants is largely unaffected. (I) A 4 dpf animal with ectopically located neuromasts of the posterior lateral-line organ was produced by coinjection of an antisense morpholino against endogenous sdf1a and a DNA construct for expression of a sdf1b-GFP fusion protein. When treated with 4-Di-2-ASP, the animal displays functional hair cells (arrowheads); note the absence of neuromasts along the trunk of the fish. Neuromasts of the anterior lateral-line organ appear not to be affected by the treatment. Some sdf1b-GFP-expressing cells (green) can still be seen at this stage (broken circle). (J) One such neuromast located over the yolk sac is labeled for actin (red) and acetylated tubulin (green) to demonstrate, respectively, hair bundles and axonal tracks. (K) A higher-magnification view of this neuromast reveals that hair cells are aligned parallel with the axonal tracks. Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)

Figure 7 Quantification of Neuromast Orientation along the Anteroposterior Axis and the Trajectory of First-Primordial Cells (A) Two histograms indicate the frequencies of neuromast orientations relative to the anteroposterior axis (left) and to axonal tracks (right). In wild-type larvae (blue), the axonal tracks are directed along the anteroposterior axis, and neuromasts are oriented parallel with both. The neuromast distributions with respect to the axis and to the tracks differ significantly from chance by a χ-square goodness-of-fit test (p < 10−200 and p < 10−50, respectively). For slow muscle omitted mutants (red), the neuromast orientation is essentially random with respect to the main body axes (p = 0.7), but largely accords with the direction of growth of the associated nerve fibers (p < 10−9). (B) Histograms compare the frequencies of neuromast orientations for slow muscle omitted (left) and fused somites (right) mutant fish. For mutants of both types, neuromasts are less frequently aligned with the anteroposterior body axis (purple) than with the axonal tracks (orange). By a χ-square test for homogeneity of categorical data, the distributions with respect to the anteroposterior axis and to the axonal tracks differ significantly (p < 10−4 and p < 0.002, respectively). Developmental Cell 2004 7, 401-412DOI: (10.1016/j.devcel.2004.07.018)