Architecture of the lateral line in larval zebrafish.

(A–B) Apical aspect of a horizontal neuromast labeled with fluorescent phalloidin (panel A) and its schematic representation (panel B). These images highlight epithelial planar polarization. Hair cell orientation is indicated by an eccentric black dot. In panel B, blue hair cells are rcHCs and green are crHCs. Grey indicates a pair of newborn hair cells whose polarity is not yet evident. (C) Drawing of a larval zebrafish in which double-point arrows indicate the global orientation of horizontal and vertical neuromasts of the posterior lateral line. (D) Simplified scheme of the selective innervation of rcHCs (light blue) by rcLANs (blue) and crHCs (green) by crLANs (red). Polarity-selective synaptogenesis renders LANs tuned to the direction of mechanical stimulation of HCs, and form labeled lines of mechanosensory flow from the periphery to the brain. Each cell is meant to represent the entire population of identically oriented HCs in the neuromast and identically tuned LANs. (E) Confocal image of a lateral view of the trunk of a double-transgenic Tg[pou4f3:GAP-GFP; hsp70l:mCherry-2.0cntnap2a] larval zebrafish, showing LANs (red) and HCs (green). (F) Magnified frontal view of a neuromast of the transgenic line Tg[myo6b:actb1-EGFP] showing HCs (green). Inset at the bottom left shows the planar polarization of the hair cells. A single LAN (red) was marked by mosaic expression of mCherry. (G) Shows a higher magnification of the inset in panel F, in which the HCs that were innervated by the single mCherry-expressing LAN in panel F are marked with a red asterisk. In this example, 7 out of the 8 constituent crHCs were innervated by this identified LAN, and none of the rcHCs were synapsed by the same neuron. Scale bars are 10 μm. In all the panels and figures, dorsal is up and anterior is left. crHC, caudorostral HC; crLAN, caudorostral LAN; HC, hair cell; LAN, lateralis afferent neuron; rcHC, rostrocaudal HC; rcLAN, rostrocaudal LAN.

Live imaging of synaptogenesis in neuromasts.

(A–B) Apical view of hair cells (green) in a horizontal neuromast of Et(krt4:EGFP)sqet4 transgenics labeled with DiAsp (red), which only penetrates into mature hair cells through mechanotransducing channels. The image reveals basal projections (white arrowheads) in 2 of the 4 immature hair cells that are DiAsp(−) (white asterisks in panel B). (C) Confocal image of a neuromast in triple-transgenics Tg[pou4f3:GAP-GFP; pou4f3:ctbp2l-mKOFP; nkhgn39d], showing juxtaposition of LAN neurites (red) with active-zone (Ribeye[+] puncta, light blue) in hair cells (green). (D–G’”) Selected time points from live imaging of innervation of hair cells (green) expressing Ribeye-Kusabira (light blue) by a singly marked LAN expressing mCherry (red), in double transgenics Tg[pou4f3:GAP-GFP; pou4f3:ctbp2l-mKOFP]. These panels were extracted from S1 Video. Ribeye(+) puncta are readily evident in hair cells (arrows) but absent from basal projections of hair cells (arrowheads in panel D–E’”). Synapses occur when LAN neurites and Ribeye(+) puncta are persistently juxtaposed over time (panel F–G’”). Dotted line outlines the hair cells. (H) Lateral view of a transgenic Tg[nkhgn39d] larval zebrafish expressing EGFP (green) in all LANs and mosaic expression of mCherry (red) in a single LAN, whose cell body (empty arrowhead) can be seen within the posterior lateralis ganglion. A neuromast (arrow) and the site of the eventual severing of the LAN peripheral axons (solid arrowheads) are indicated. The red rhomboid below the indicated neuromast is a singly marked myofiber resulting from nonspecific expression of mCherry in muscle. (I–K) Magnified views of the same fish in panel H, showing the LAN peripheral axons before laser-mediated severing (panel I), immediately after severing (panel J), and several hours after severing (panel K), evidencing the separation of the proximal and distal parts of the axon fragments. The arrows indicate the same neuromast and the solid arrowheads the site of the cut in every panel. The red rhomboid is the same myofiber in panel H. (L–O) Selected time points from live confocal imaging of regenerative innervation of mature hair cells (green) by the singly marked LAN (red). Panels were generated from S2 Video. Hair cells never produce basal projections during polarity-selective re-innervation. Scale bars are 10 μm in panel A, C, and D and 50 μm in panel I. DiAsp, 4-(4-diethy-laminostyryl)-N-methylpyridinium iodide; EGFP, enhanced green fluorescent protein; HC, hair cell; LAN, lateralis afferent neuron; Rib-Kus, Ribeye-Kusabira.

Regenerative synaptogenesis in neuromasts.

(A–C) A neuromast of the transgenic line Tg[myo6b:actb1-EGFP; nkhgn39d] with mosaic expression of mCherry in a single LAN. (A) The individualized axon (red) synapses with crHCs. (A’) Magnified view of the hair bundles of hair cells in panel A. (B) Schematic representation of the example in panel A, in which hair cell orientation is indicated by an eccentric black dot and innervation by a red asterisk. (C) After severing, the individualized regenerated axon (red) recapitulate synapses with crHCs in the same neuromast. (C’) Magnified view of the hair bundles of hair cells in panel C. (D) Schematic representation of the example in panel C, indicating hair cell orientation (eccentric black dot) and synapses (red asterisk). (E) Neuromast double-transgenic Tg[myo6b:actb1-EGFP; Ribeye-Kusabira] showing a single LAN marked by mosaic expression of mCherry. The axon (red) synapses with rcHCs. (E’) Magnified view of the hair bundles of hair cells in panel E. (F) Schematic representation of the example in panel E, in which hair cell orientation is indicated by an eccentric black dot and innervation by a red asterisk. (G) After severing, the individualized axon (red) regenerated to recapitulate synapses with rcHCs. (G’) Magnified view of the hair bundles of hair cells in panel G. (H) Schematic representation of the example in panel G, indicating hair cell orientation (eccentric black dot) and synapses (red asterisk). (I) Quantification of hair cell innervation by regenerating singly marked axons of samples in which the axonal bundle was severed immediately below the neuromast (N = 35) (near neuromast) or furthest (N = 18) (near the posterior lateralis ganglion). All 35 axons cut near neuromast re-innervated hair cells of the original orientation—but 16 of the axons cut near the ganglion (0.89)—re-innervated the original orientation after regeneration (memory in grey), and 2 (0.11) did not (no memory in pink). (J) Quantification of neuromast innervation by regenerating singly marked axons of samples in which the lateralis nerves were severed immediately below the neuromast (near neuromast) (N = 35) or furthest (near the ganglion) (N = 18). A total of 23 (0.66) individualized axons cut near neuromast re-innervated the original neuromast (memory in grey), whereas 12 (0.34) re-innervated a different organ (no memory in pink). When axons were cut near the ganglion, 4 (0.22) re-innervated the original neuromast (memory in grey), and 14 (0.78) re-innervated a different organ (no memory in pink). (K–O”) Neuromast of the transgenic line Tg[myo6b:actb1-EGFP; nkhgn39d] with mosaic expression of mCherry in a single LAN. (K–K’) individualized axon (red) synapse with rcHCs. (K’) Magnified view of the hair bundles of hair cells in panel K. (L) Schematic representation of the example in panel K. (M) After axon severing and hair cell elimination, the individualized axon (red) regenerated to synapses with regenerated rcHCs in the same neuromast. (M’) Magnified view of the hair bundles of hair cells in panel M. (N) Schematic representation of the example in panel L–M. (O–O”) Selected time points from live confocal imaging of regenerative innervation of hair cells in the transgenic line Tg[myo6b:actb1-EGFP; nkhgn39d] (green) in an instance when a singly marked LAN (red) switched from a horizontal neuromast (solid arrowhead) to a vertical neuromast (empty arrowhead). Before laser-mediated severing (panel O), after severing (panel O’), and after regeneration (panel O”). The white arrow indicated the site of the cut. (P–P’, R–R’) Neuromast of the transgenic line Tg[myo6b:actb1-EGFP; nkhgn39d] with mosaic expression of mCherry in a single LAN. (P) The individualized axon (red) synapses with crHCs in a horizontal neuromast. (P’) Magnified view of the hair bundles of hair cells in panel P. (Q) Schematic representation of the example in panel P. (R) After severing, the individualized regenerated axon (red) switches to innervate a vertical neuromast, in which it synapses selectively with vdHCs. (R’) Magnified view of the hair bundles of hair cells in panel R. (S) Schematic representation of the example in panel R. Scale bars are 10 μm in A and A’ and 50 μm in O. crHC, caudorostral HC; HC, hair cell; LAN, lateral afferent neuron; rcHC, rostrocaudal HC; vdHC, ventrodorsal HC.

Emx2 expression in larval neuromasts.

(A–C) Immunohistochemical staining of a horizontal neuromast in the transgenic line Tg[myo6b:actb1-EGFP] showing HCs (green) and Emx2 (red, dotted circles). Inset in panel B is a superficial image of the hair cells’ apices revealing their planar polarization. (D) Scheme of the neuromast in panel A–C indicating Emx2-expressing (red) and nonexpressing (green) hair cell. (E–G) Immunohistochemical staining of a vertical neuromast in the transgenic line Tg[myo6b:actb1-EGFP] showing HCs (green) and Emx2 expression (red, dotted circles). Inset in panel F is a superficial image of the hair cells’ planar polarization. (H) Scheme of the neuromast in panel E–G indicating Emx2-expressing (red) and nonexpressing (green) hair cell. (I–K) Immunohistochemical staining of a horizontal neuromast in the transgenic line Tg[myo6b:actb1-EGFP] showing HCs (green) (panel I), a single LAN expressing mCherry (red) (panel J). White asterisks indicate Emx2-expressing hair cells (purple) (panel K). Inset in panel I reveals hair cell planar polarization. (L) Scheme of the neuromast in panel I–K indicating Emx2-expressing hair cell (purple), nonexpressing hair cell (green), and the synapse of the individualized axon (red asterisks). The polarity of hair cell could not be determined (grey). In panel B, F, I, and N, neuromast orientation is indicated by a double-head arrow. (M–O) Staining of a horizontal neuromast in a neurogenin1 mutant specimen, with an antibody to Emx2 and phalloidin to reveal hair cell apices. It shows Emx2-expressing cells (green, dotted circles) and hair cell orientation (panel N) (red, double-head arrow) (panel N). (P) Scheme of the neuromast in panel M–O indicating Emx2-expressing (red) and nonexpressing (green) hair cell, indicating hair cell orientation (eccentric black dots). Scale bars are 10 μm. HC, hair cell; LAN, lateralis afferent neuron.

Regenerative synaptogenesis of neuromasts.

(A–B) Apical aspect of a neuromast in Vangl2 mutant, labeled with fluorescent phalloidin (panel A) and its schematic representation (panel B). These images highlight the loss of coherent epithelial planar polarity in the neuromast. (C–D) Immunohistochemical staining of a horizontal neuromast in the transgenic line Et(krt4:EGFP)sqet4 mutant for Vangl2 showing HCs (panel C) and Emx2 (panel D). Dotted circles indicate Emx2(+) cells. (E) Quantification of Emx2 expression in wild-type (blue) and Vangl2 mutant (red) neuromasts. Numbers on the y-axis indicate the fraction (ratio) of the hair cells expressing Emx2; one-way ANOVA. Error bars indicate mean ± SD. (F–K) A Vangl2−/− neuromast expressing EGFP in hair cells (panel F), immunostained for Emx2 (panel G), labeled with 488-phalloidin to reveal planar polarity (panel J) and expressing mCherry in a single LAN (panel H–I). Emx2 is expressed in 3 out of 7 hair cells (dashed circles) (panel G). A purple star marks an Emx2(+) cell that is not a hair cell (panel F–G). The marked LAN (panel H–I) innervates the 4 Emx2(−) hair cells as shown in the scheme (panel K), which is color-coded (blue for Emx2[+] and grey for Emx2[−]). (L) Quantification of the number of hair cells innervated by an identified axon in horizontal neuromasts in wild-type specimens (blue) and in those lacking Vangl2 (green) or Emx2 (orange/red/violet). Emx2 mutant neuromast were separated into those containing 0 rcHCs (orange), 1 rcHC (red), and 2 rcHCs (violet). Numbers on the y-axis indicate the fraction (ratio) of innervated hair cells. ***p < 0.001; one-way ANOVA. Error bars indicate mean ± SD. (M–N) A neuromast in the transgenic line Et(krt4:EGFP)sqet4 expressing mCherry in a single LAN, showing HCs (panel M) and axonal terminal arborization (panel N). (O) A scheme of panel M–N indicating hair cell orientation (eccentric black dots) and the synapse of the individualized axon (red asterisks). (P–Q) A vertical (panel P) and horizontal (panel Q) neuromast from specimens lacking Emx2, showing the typical homogeneous polarization of hair cells. (R–S) A vertical neuromast lacking Emx2 in the transgenic line Tg[pou4f3:GAP-GFP; pou4f3:ctbp2l-mKOFP] expressing mCherry in a single LAN, showing juxtaposition of LAN neurites (red) with the active-zone Ribeye(+) puncta (yellow) (panel R), and presence of Ribeye(+) puncta (yellow) in hair cells (green) (panel S). Dotted line indicates the hair cells. (T) A scheme of panel R–S indicating hair cell orientation (eccentric black dots) and the synapse of the individualized axon (red asterisks). Note that the noninnervated hair cells are Ribeye(−) and therefore are likely immature. (U–V) A horizontal neuromast lacking Emx2 in the transgenic line Tg[pou4f3:GAP-GFP] expressing mCherry in a single LAN, showing no synapse of the LAN with any of the hair cells. Dotted line indicates the hair cells. (W) A scheme of panel U–V showing hair cell orientation (eccentric black dots). Scale bars are 10 μm. EGFP, enhanced green fluorescent protein; Emx2,; HC, hair cell; LAN, lateralis afferent neuron; ns, not significant; rcHC, rostrocaudal HC; Vangl2,; wt, wild-type.