Supplementary MaterialsS1 Fig: Tmie-GFP shows variable expression in stereocilia. region, in B and E is the whole hair cell, and in C and F is definitely a subtraction of whole cell fluorescence minus soma fluorescence to roughly determine the relative contribution of package signal. Significance was determined by two-tailed unpaired t-test with Welchs correction, **p 0.01, ****p 0.0001.(TIF) pgen.1007635.s002.tif (1.0M) GUID:?C7360E0A-46DD-4A5B-AA3C-A6AE3D647BC4 S3 Fig: Differential effects on function having a genomic mutation and a transgene CLEC4M mimic. (A) Data for any novel mutant allele of (below) showing the genomic region where the mutation happens. An arginine is definitely mutated to guanine in the splice acceptor (black package, above) of the final exon of larvae bridging exons 3 and 4. Protein: The predicted protein products, shown here as a two-pass transmembrane protein. The wild type protein has many charged residues (positive in light gray, negative in dark gray) that are lost in larvae, taken with a hand-held Canon camera. Arrow points to a larva that is upside-down, displaying a classic vestibular phenotype. (B) Top-down view of a representative neuromast after MK-4305 cost exposure to FM 4C64, imaged using confocal microscopy. The first panel is a single plane through the soma region while the second panel is a maximum projection of 7 panels through the soma region, beginning at the cuticular plate (as denoted by magenta bracket in Fig 1G). (C) Same as (B) except that the first panel shows the bundle region so that 1-138-GFP can be visualized in bundles (as depicted by dashed green line, Fig 1G). The transgene is driven by the promoter. (D) Plot of the integrated density of FM fluorescence per cell. We normalized values to the average of wild type siblings. Displayed wild type and data are from siblings of and are the same values reported in Fig 6. Data for is from a separate experiment. Statistical significance determined by one-way ANOVA, ****p 0.0001. Scale bar is 10m.(TIF) pgen.1007635.s003.tif (3.5M) GUID:?7AAA631D-EEC2-4675-831E-68A6DE67C18C S4 Fig: Expression pattern and functional rescue by constructs CD8 and 139C231. All images were captured using confocal microscopy. (A) Stereocilia of a neuromast viewed from above. The same neuromast was imaged at 4 dpf and 6 dpf. In hair cells expressing CD8-GFP, signal was initially detected in immature bundles, but this expression was only detectable in soma by dpf 6 as the cells matured (n = 10 cells). (B) Maximum projection of neuromasts viewed from above; left panel shows only MK-4305 cost FM 4C64 while right panel adds CD8-GFP. No rescue of FM 4C64 labeling was observed in hair cells expressing CD8-GFP (n = 40 cells). (C) Maximum projection of the posterior crista in a larva with some hair cells expressing 139-231-GFP, which fills the cell (n = 43 cells). (D) Same as B except the transgene becoming expressed can be 139-231-GFP. No save of FM 4C64 labeling was seen in locks cells expressing 139-231-GFP (n = 33 cells). Size pubs in C and A are 5m, in D and B are 10m.(TIF) pgen.1007635.s004.tif (3.7M) GUID:?71A1D86D-24FB-4014-84D0-3E7A8465547C S5 Fig: Nuclear mCherry fluorescence will not correlate with GFP-tagged Tmc fluorescence. XY plots from the integrated denseness of nuclear mCherry fluorescence vs the integrated denseness of GFP-tagged Tmc fluorescence in the package area of lateral cristae. We analyzed 4 dpf larvae. (A) Package ideals for constructs Compact disc8-2TM and 97C113 will be the identical to those reported in Fig 8H using co-expression with Tmc2b-GFP. Package ideals for the MK-4305 cost full-length Tmie create are the identical to those reported in Fig 4C using co-expression with Tmc1-GFP. (B) Package values will be the identical to.