smpx expression in the neuromasts of the posterior lateral line. Representative stereomicroscope images of in-situ hybridizations performed with the smpx riboprobe on whole mount larvae at 96 and 120 hpf. (A,A’) The gene is expressed in all the neuromasts (L1-L7, black arrowheads) establishing the lateral line at both stages analyzed. The smpx-specific signal is present also in the accessory neuromasts (green arrowheads) and in the terminal neuromasts of the tail (black arrows). (B,B’) Detailed view of smpx signal in the L2, L3 and L6 neuromasts of the trunk at 96 and 120 hpf. The white-dotted rectangles show magnified neuromasts with the typical rosette-like structure, the green arrowheads indicate the accessory neuromasts. (C,C’) Detailed view of smpx signal in the L6, L7, and terminal neuromasts (black arrows) of the tail at 96 and 120 hpf. (A,B) Neuromasts appear slightly below the median line because they were flat-mounted under a coverslip. Lateral views, anterior to the left. Scale bars are 160 µm.

Smpx protein localization in the neuromast mechanosensory hair cells. Confocal Z-stacks of immunofluorescence on whole mount 120 hpf larvae. (A–C) Smpx (green) localizes in the most internal cells of the neuromast, namely the mechanosensory hair cells, while it is excluded from the outermost cells (red) of the transgenic line tg (sod1:sod1; hsp70:DsRed) and the presumptive accessory cell layers (white bracket) encompassed in between the outmost DsRed-positive cells and the inmost hair cells. (D–F) Smpx (red) co-localizes with acetylated tubulin (green) in the kinocilium, with Smpx showing a ‘beads-on-a-string’ distribution (arrowheads in D). In some cases, the kinocilium is not labeled by the anti-Smpx antibody (arrow in F). (A–F) Larvae were counterstained with DAPI (blue) to visualize cell nuclei. Lateral views, anterior to the left. Scale bars are 15 µm in A–C and 10 µm in D–F.

Smpx-deficiency leads to a lower number of intact neuromasts in morphant and crispant larvae. (A) Quantification of the reduced number of intact neuromasts in smpxMO versus ctrlMO larvae. (B,C) Representative confocal images of ctrlMO and smpxMO larvae labeled with an anti-acetylated tubulin antibody (magenta). Larvae were counterstained with DAPI to visualize cell nuclei. Insets in C show magnified neuromasts lacking the innermost nuclei and the knocilia of the HCs (compare to inset in B). (D) Quantification of the reduced number of intact neuromasts in smpx^crispant versus ctrl larvae. (E,F) Representative confocal images of immunofluorescence experiments with an anti-GFP antibody in ctrl and smpx^crispanttg (cldnb:GFP) transgenic larvae. Insets in F display magnified neuromasts showing the depletion of the innermost cell cluster (compare to inset in E). (A,D) Unpaired t-test was performed to assess statistical significance by setting p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****) as significant. (A) ctrlMO n = 8, smpxMO n = 8. (D) ctrl n = 19, smpx^crispant n = 22. (B,C and E,F) Confocal Z-stacks images taken from 120 hpf whole mount larvae; lateral views, anterior to the left. Scale bars are 500 µm.

Smpx-deficiency causes the decrease in the PLLp size. (A,B) Immunofluorescence with the anti-Smpx antibody unveiling the localization of the protein in the PLLp at 26 and 29 hpf. (C,D) In-situ hybridization with a prox1a probe to compare the PLLp size (white-dotted encircled areas) in 30 hpf ctrlMO and smpxMO; the PLLp of Smpx-deficient embryos appears smaller in size; the typical rosette-like structures present in the ctrlMO sample (white arrowheads in C) are not clearly distinguishable in the smpxMO sample. (E) Size quantification in ctrlMO and smpxMO showing a drastic decrease in the PLLp area following the downregulation of smpx. (F,G) In-situ hybridization with a prox1a probe to compare the PLLp size (white-dotted encircled areas) in 30 hpf ctrl and smpx^crispant embryos; the PLLp of the smpx^crispant embryos appears smaller in size and the typical rosette-like structures present in the ctrl sample (white arrowheads in F) are not clearly distinguishable in the smpx^crispant preparation. (H) Size quantification in ctrlMO and smpx^crispant showing a drastic decrease in the PLLp area following the ablation of smpx. (I,J) Z-stack confocal images of the PPLp at 30 hpf in tg (cldnb:GFP) embryos; the shape of the epithelial rosettes in control embryos are lost in smpx^crispants. (A,B) Confocal Z-stacks taken from whole mount embryos; lateral views, anterior to the left. Embryos were counterstained with DAPI to visualize cell nuclei. (E,H) Unpaired t-test was performed to assess statistical significance by setting p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****) as significant. (E) ctrlMO n = 22, smpxMO n = 18. (H) ctrl n = 10, smpx^crispant n = 13. Scale bars are 50 µm.

Smpx-deficiency causes a reduced mechanotransduction activity of the neuromast hair cells. (A–C) Representative images of FM 4–64 incorporation in one control neuromast (ctrlMO) and two Smpx-deficient (smpxMO) neuromasts at 120 hpf (white rectangles on the right of each single image are magnifications of the corresponding, white-dotted rectangles on the left); compared to the control (A), smpx-morphants (B,C) display a reduced FM 4–64 dye uptake. (D) Quantification of the FM 4–64 dye uptake calculated as fluorescence intensity per each neuromast (n = 10 for both controls and morphants). (A–C) Confocal Z-stacks taken from whole mount larvae. (D) Unpaired t-test was performed to assess statistical significance by setting p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***) and p ≤ 0.0001 (****) as significant. Scale bars are 50 µm.

Smpx is required for normal kinocilium structure. Immunofluorescence performed on whole mount 120 hpf larvae with an antibody against acetylated tubulin to label the neuromast kinocilia. (A,B) Compared to controls (ctrlMO), the kinocilia of morphant larvae (smpxMO) appear clearly shorter in length and numerically less dense. (C,D) As for the knockdown experiment, when compared to controls (ctrl), the kinocilia of crispant larvae (smpx^crispant) are shorter and numerically lower. (A,B) ctrlMO n = 5, smpxMO n = 4. (C,D) ctrlMO n = 5, smpxMO n = 5. (A–D) Confocal Z-stack images of neuromast L4 for each larva. n is equal to the sum of the larvae from two independent experiments. Embryos were counterstained with DAPI to visualize cell nuclei. Scale bars are 6 µm.

smpx shapes the lateral line organ and affects its mechanosensitivity. Smpx-deficiency affects the number of intact neuromasts in the PLL, possibly because of the role of the gene in directing the development of the HCs in the PLLp. Furthermore, Smpx is essential in proper kinocilium modeling and HCs mechanotransduction.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.