( C ) The autocorrelation was used to interpret the burst duration and burst frequency changes . Burst duration is measured by the intersect of the two linear fits . When burst duration is constant , the amplitude is inversely related to the burst frequency . ( D ) Burst duration from autocorrelation is constant across 4 different galactose concentrations ( 2 % : n = 86 cells , 0 . 2 % : n = 79 cells , 0 . 02 % : n = 84 cells , 0 . 004 % : n = 53 cells ) . Error bars indicate SEM . ( E ) Amplitude of autocorrelation decreases ( burst frequency increases ) at higher doses of galactose . ( 2 % : n = 86 cells , 0 . 2 % : n = 79 cells , 0 . 02 % : n = 84 cells , 0 . 004 % : n = 53 cells ) . Error bars indicate SEM .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The autocorrelation was used to interpret the burst duration and burst frequency changes. Burst duration is measured by the intersect of the two linear fits. When burst duration is constant, the amplitude is inversely related to the burst frequency. (D) Burst duration from autocorrelation is constant across 4 different galactose concentrations (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM. (E) Amplitude of autocorrelation decreases (burst frequency increases) at higher doses of galactose. (2%: n = 86 cells, 0.2%: n = 79 cells, 0.02%: n = 84 cells, 0.004%: n = 53 cells). Error bars indicate SEM.


( F ) Example traces of two single cells exposed to two different doses of galactose . ( G , H ) Same - cell dose response shows same burst duration ( G ) but lower amplitude ( H ) at higher galactose concentration , indicating that galactose levels regulate burst frequency ( n = 13 cells ) . Error bars indicate SEM .

O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(F) Example traces of two single cells exposed to two different doses of galactose. (G, H) Same-cell dose response shows same burst duration (G) but lower amplitude (H) at higher galactose concentration, indicating that galactose levels regulate burst frequency (n = 13 cells). Error bars indicate SEM.


A , B . Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos . Bead tracks every 500 ms over 2 s are shown ( A ) . Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants , which can be fully rescued by co - injecting with Xenopus ccdc108 mRNA ( B ) . 150 beads from 15 embryos for each condition . Arrowheads indicate beads that showed no movement on the embryo surface through the recording . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Depletion of Ccdc108 markedly affects bead movement by decreasing velocity Fluorescent beads were applied to the dorsal surface of Xenopus embryos. Bead tracks every 500 ms over 2 s are shown (A). Plot of average bead velocity shows significantly reduced bead flow in ccdc108 morphants, which can be fully rescued by co-injecting with Xenopus ccdc108 mRNA (B). 150 beads from 15 embryos for each condition. Arrowheads indicate beads that showed no movement on the embryo surface through the recording. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


C , D . Defective cilia formation in ccdc108 morphants . Embryos at stage 27 were fixed and stained with the acetylated tubulin ( Ac - tub ) antibody and / or Cep164 antibody . mRNA of a membrane - bound form of GFP ( mGFP ) was co - injected with each morpholino to indicate targeted cells . Representative confocal images ( C ) and 3D - SIM images ( D ) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos . ( C ) 20 images of 20 embryos and ( D ) > 80 MCCs from 6 embryos for each condition . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

O O O O O B-SUBCELLULAR O O B-GENEPROD O O B-TISSUE O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-TISSUE O O O O O O O O B-TISSUE O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Defective cilia formation in ccdc108 morphants. Embryos at stage 27 were fixed and stained with the acetylated tubulin (Ac-tub) antibody and/or Cep164 antibody. mRNA of a membrane-bound form of GFP (mGFP) was co-injected with each morpholino to indicate targeted cells. Representative confocal images (C) and 3D-SIM images (D) of the epidermis for each condition and plots show significantly reduced cilia number in Ccdc108 depleted embryos. (C) 20 images of 20 embryos and (D) > 80 MCCs from 6 embryos for each condition. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


F . Defective cilia formation occurs in ccdc108 CRISPR mutants . Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 ( 108sg ) and fixed at stage 27 . Representative 3D - SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants . > 70 MCCs from 6 embryos for each condition . Cell membranes ( mGFP , purple ) , cilia ( Ac - tub , green ) and basal bodies ( Cep164 , yellow ) were labeled with indicated antibodies . Data information : Quantification data were collected from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are presented .

O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD O O O O B-CELL_TYPE O O B-TISSUE O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Defective cilia formation occurs in ccdc108 CRISPR mutants. Embryos at one cell stage were injected with Cas9 protein with or without the sgRNA against ccdc108 (108sg) and fixed at stage 27. Representative 3D-SIM images and plot show significantly reduced cilia number in ccdc108 CRISPR mutants. > 70 MCCs from 6 embryos for each condition. Cell membranes (mGFP, purple), cilia (Ac-tub, green) and basal bodies (Cep164, yellow) were labeled with indicated antibodies. Data information: Quantification data were collected from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are presented.


G . In situ hybridization chain reaction ( HCR ) reveals that ccdc108 mRNA is specially detected in MCCs . Embryos at stage 35 were fixed and subjected to in situ HCR ( red ) . Embryos were then incubated with the Ac - tub antibody ( cyan ) and DAPI ( gray ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-CELL_TYPE O B-TISSUE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O

G. In situ hybridization chain reaction (HCR) reveals that ccdc108 mRNA is specially detected in MCCs. Embryos at stage 35 were fixed and subjected to in situ HCR (red). Embryos were then incubated with the Ac-tub antibody (cyan) and DAPI (gray).


C , D . Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts ( C ) and olfactory placodes ( D ) . Zebrafish embryos were fixed and stained with the Ac - tub antibody ( green ) , phalloidin ( purple ) and Hoechst ( blue ) . Data information : Quantitative data were from three independent repeats . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . Mean ± s . e . m values are presented .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O B-EXP_ASSAY O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O B-ORGANISM B-TISSUE O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Representative confocal images and plots show that ccdc108 morphants displayed ciliogenesis defects in neuromasts (C) and olfactory placodes (D). Zebrafish embryos were fixed and stained with the Ac-tub antibody (green), phalloidin (purple) and Hoechst (blue). Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). Mean ± s.e.m values are presented.


E . Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O B-SUBCELLULAR O O B-GENEPROD O B-TISSUE I-TISSUE O

E. Scanning electron microscope images of zebrafish embryos display a failure in cilia formation in Ccdc108 depleted olfactory placodes.


H . Representative 3D - SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs . mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining . GFP - CETN1 ( blue ) marked mEPCs infected with lentivirus . Basal bodies ( CEP164 , purple ) and cilia ( Ac - tub , green ) were labeled with indicated antibodies . > 60 MCCs from three independent repeats for each condition were counted . Data information : Quantitative data were from three independent repeats . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * p < 0 . 05 ) . mean ± s . d . values are presented .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O B-ORGANISM O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative 3D-SIM images and plot reveal that depletion of CCDC108 leads to a significant reduction in multiciliogenesis in mEPCs. mEPCs infected with lentivirus expressing indicated shRNA were serum starved for five days and subjected to immunostaining. GFP-CETN1 (blue) marked mEPCs infected with lentivirus. Basal bodies (CEP164, purple) and cilia (Ac-tub, green) were labeled with indicated antibodies. > 60 MCCs from three independent repeats for each condition were counted. Data information: Quantitative data were from three independent repeats. Unpaired two-tailed t-test was performed (*** p<0.001; * p<0.05). mean ± s.d. values are presented.


A . Depletion of Ccdc108 causes disorganized basal body distribution in MCCs . Representative 3D - SIM images ( x - y ) and 3D - reconstructions ( x - z ) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells . Cell membranes ( mGFP , blue ) , basal bodies ( Cetn1 , green ) and distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations . Orange arrowheads and numbers mark individual centriole . > 70 MCCs from 6 embryos for each condition were counted . Data information : Quantitative data were from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_TYPE O B-TISSUE O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Depletion of Ccdc108 causes disorganized basal body distribution in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs of embryos show a failure of apical trafficking of basal bodies in Ccdc108 depleted cells. Cell membranes (mGFP, blue), basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Select area of Cep164 channel was zoomed to show centriolar Cep164 accumulations. Orange arrowheads and numbers mark individual centriole. > 70 MCCs from 6 embryos for each condition were counted. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


B . Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs . Orange arrowheads mark centrioles . Percentage of centrioles docked to plasma membrane in each field were scored . At least 7 cells for each sample were counted .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O

B. Transmission electron microscopy images of Xenopus embryos displaying aberrant apical trafficking and docking of centrioles in Ccdc108 depleted MCCs. Orange arrowheads mark centrioles. Percentage of centrioles docked to plasma membrane in each field were scored. At least 7 cells for each sample were counted.


C . Representative 3D - SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs . Embryos were fixed and stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Apical area of MCCs in each condition were measured ; 80 MCCs from 6 embryos for each condition . D . Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108 . Cell boundaries and apical actin network were marked with mGFP ( green ) and phalloidin ( purple ) and phalloidin intensity levels measured and plotted . > 80 MCCs from 6 embryos for each condition . Data information : Quantitative data were from three independent experiments . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ; * p < 0 . 05 ) . Mean ± s . d . values are presented .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-CELL_TYPE O B-TISSUE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Representative 3D-SIM images reveal that depletion of Ccdc108 has no effect on apical expansion in MCCs. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Apical area of MCCs in each condition were measured ; 80 MCCs from 6 embryos for each condition. D. Representative confocal images show a significant reduction in apical actin following depletion of Ccdc108. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple) and phalloidin intensity levels measured and plotted. > 80 MCCs from 6 embryos for each condition. Data information: Quantitative data were from three independent experiments. Unpaired two-tailed t-test was performed (*** p<0.001 ; ** p<0.01; * p<0.05). Mean ± s.d. values are presented.


A . Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies . Embryos expressing GFP - Ccdc108 ( green ) and Cetn4 - RFP ( purple ) were imaged at stage 27 . To better visualize ciliary structures , maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O

A. Live imaging of MCCs shows the distribution of Ccdc108 in MCCs cilia and basal bodies. Embryos expressing GFP-Ccdc108 (green) and Cetn4-RFP (purple) were imaged at stage 27. To better visualize ciliary structures, maximum intensity projections of images stacks for regions containing cilia and basal bodies are shown separately.


C . Ccdc108 interacts with IFT proteins . Interactions were further determined by coimmunoprecipitation of endogenous IFT proteins from HEK293T cells with GFP - Trap agarose beads .

O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-CELL_LINE O O B-GENEPROD O O O O O

C. Ccdc108 interacts with IFT proteins. Interactions were further determined by coimmunoprecipitation of endogenous IFT proteins from HEK293T cells with GFP-Trap agarose beads.


D . Live imaging of MCCs shows both Ccdc108 and Ift74 proteins localize to the ciliary axoneme and the basal bodies in MCCs . Embryos expressing mCherry - Ccdc108 ( green ) and GFP - Ift74 ( purple ) were imaged at stage 27 .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-TISSUE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

D. Live imaging of MCCs shows both Ccdc108 and Ift74 proteins localize to the ciliary axoneme and the basal bodies in MCCs. Embryos expressing mCherry-Ccdc108 (green) and GFP-Ift74 (purple) were imaged at stage 27.


A - C . The IFT - interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos : ( A ) impaired multiciliogenesis , ( B ) defective apical migration / docking of centrioles , and ( C ) reduced enrichment of apical F - actin . Embryos at stage 27 were fixed and stained with the acetylated tubulin ( A ; Ac - tub ; green ) , Cep164 ( B ; purple ) and Cetn1 ( B ; green ) antibodies or phalloidin ( purple ) . mRNA of a membrane - bound form of GFP was co - injected with each morpholino to indicate targeted cells ( A , B ) . Typical D - SIM images ( A , B ) and confocal images ( C ) are present . Embryos were treated as described in Figure 1C and expressed mGFP or GFP - Ccdc108 mutants . Plots show results from three independent experiments . ( A ) > 70 MCCs from 6 embryos , and ( C ) > 70 MCCs from 6 embryos for each condition . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are presented .

O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. The IFT-interaction domain is required for Ccdc108 multiciliogenesis function in Xenopus embryos: (A) impaired multiciliogenesis, (B) defective apical migration/docking of centrioles, and (C) reduced enrichment of apical F-actin. Embryos at stage 27 were fixed and stained with the acetylated tubulin (A; Ac-tub; green), Cep164 (B; purple) and Cetn1 (B; green) antibodies or phalloidin (purple). mRNA of a membrane-bound form of GFP was co-injected with each morpholino to indicate targeted cells (A,B). Typical D-SIM images (A,B) and confocal images (C) are present. Embryos were treated as described in Figure 1C and expressed mGFP or GFP-Ccdc108 mutants. Plots show results from three independent experiments. (A) > 70 MCCs from 6 embryos, and (C) > 70 MCCs from 6 embryos for each condition. Unpaired two-tailed t-test was performed (*** p<0.001; **p<0.01). Mean ± s.d. values are presented.


A , ift74 CRISPR mutants display defective cilia formation . Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74 . Representative confocal images of the Xenopus epidermis , and plots display defective ciliation . ; 60 MCCs from 6 embryos Cell membranes ( mGFP ) , cilia ( Ac - tub , green ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O O O O O O O B-CELL_TYPE O O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative confocal images of the Xenopus epidermis, and plots display defective ciliation. ;60 MCCs from 6 embryos Cell membranes (mGFP), cilia (Ac-tub, green) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


B . ift74 CRISPR mutants display defective cilia formation . Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74 . Representative 3D - SIM images of the Xenopus epidermis , and plots display defective ciliation . ; 70 MCCs from 6 embryos for each condition . Cell membranes ( mGFP ) , cilia ( Ac - tub , green ) and basal bodies ( Cep164 ) were labeled with indicated antibodies . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O B-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative 3D-SIM images of the Xenopus epidermis, and plots display defective ciliation. ;70 MCCs from 6 embryos for each condition. Cell membranes (mGFP), cilia (Ac-tub, green) and basal bodies (Cep164) were labeled with indicated antibodies. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C . Ift74 depletion affects centriole apical migration in MCCs . Representative 3D - SIM images ( x - y ) and 3D - reconstructions ( x - z ) of MCCs show failure of apical trafficking of basal bodies . Basal bodies ( Cetn1 , green ) and distal appendages ( Cep164 , purple ) were labeled with indicated antibodies . Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74 . > 70 MCCs from 6 embryos for each condition . D . Representative 3D - SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion . > 70 MCCs from 6 embryos for each condition . Embryos were fixed and stained with phalloidin ( purple ) . Cilia ( Ac - tub , gray ) were also presented . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O B-CELL_TYPE O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O B-GENEPROD O O O O B-CELL_TYPE O O B-TISSUE O O O O B-TISSUE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Ift74 depletion affects centriole apical migration in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs show failure of apical trafficking of basal bodies. Basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74. >70 MCCs from 6 embryos for each condition. D. Representative 3D-SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion. >70 MCCs from 6 embryos for each condition. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


E . Representative confocal images and plot show a significant reduction in apical F - actin enrichment upon Ift74 depletion . > 70 MCCs from 6 embryos for each condition . Cell boundaries and apical actin network were marked with mGFP ( green ) and phalloidin ( purple ) . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-CELL_TYPE O O B-TISSUE O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative confocal images and plot show a significant reduction in apical F-actin enrichment upon Ift74 depletion. >70 MCCs from 6 embryos for each condition. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple). Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C - E . Ccdc108 depletion affects centriolar distribution of IFT - B proteins during centriole migration in MCCs . Embryos were co - injected with GFP - IFTs ( C : Ift74 ; D : Ift80 ; E : Ift88 ; green ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . HA tagged Ccdc108 proteins were co - expressed to test for the rescue of phenotypic defects . Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of IFT - B proteins ( Ift74 , Ift80 and Ift88 ) , and Ccdc108 - IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT - B proteins . > 40 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ) . Mean ± s . d . values are also presented .

O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE O B-TISSUE O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E. Ccdc108 depletion affects centriolar distribution of IFT-B proteins during centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D: Ift80; E: Ift88; green), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.


A , B . Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion . Embryos were co - injected with GFP - Cp110 ( A ) GFP - Fak ( B ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . > 30 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion. Embryos were co-injected with GFP-Cp110 (A) GFP-Fak (B), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


C , D . Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs . Embryos were co - injected with GFP - Drg1 ( C ) GFP - RBD ( D ) , Cetn4 - RFP ( purple ) and each morpholino , and imaged at stage 18 . HA tagged Ccdc108 proteins were co - expressed to test for the rescue of phenotypic defects . Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels . α - tubulin ( α - Tub ) served as loading control . Quantitative plots show that Ccdc108 - depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD , and neither of Ccdc108 - IFT interaction deficient mutants can rescue the centriolar phenotypic defects . > 30 MCCs from 6 embryos for each condition . Data information : Quantitative data from three independent experiments were scored . Unpaired two - tailed t - test was performed ( * * * p < 0 . 001 ; * * p < 0 . 01 ) . Mean ± s . d . values are also presented .

O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-CELL_TYPE O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs. Embryos were co-injected with GFP-Drg1 (C) GFP-RBD (D), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD, and neither of Ccdc108-IFT interaction deficient mutants can rescue the centriolar phenotypic defects. >30 MCCs from 6 embryos for each condition. Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.


Total number of SNVs / InDels ( allele frequency ≥ 20 % ) detected in primary samples ( x - axis ) plotted against the number of SNVs / InDels detected in corresponding PDX samples ( y - axis ) ; R² coefficient of determination Number of preserved SNVs / InDels ( black bars ) and those that were not detected in PDX models ( grey bars ) in relation to their allele frequencies ( AF ) ; % - fraction of preserved

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

Total number of SNVs/InDels (allele frequency≥20%) detected in primary samples (x-axis) plotted against the number of SNVs/InDels detected in corresponding PDX samples (y-axis); R² coefficient of determination Number of preserved SNVs/InDels (black bars) and those that were not detected in PDX models (grey bars) in relation to their allele frequencies (AF); % - fraction of preserved mutations


Large ( > 1Mb ) CNA ( amplifications - shades of violet ; deletions - shades of red ) identified by low coverage whole genome sequencing in each of the patients and corresponding

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O

Large (>1Mb) CNA (amplifications- shades of violet; deletions - shades of red) identified by low coverage whole genome sequencing in each of the patients and corresponding PDXs


Allele frequencies ( AF ) of mutations detected in primary ( PRI ) patients ' samples ( x - axis ) at initial diagnosis ( INI ) and relapse ( REL ) plotted against AF of corresponding mutations detected in matched PDX models ( y - axis ) for relapse type 1 ( left ) and relapse type 2 patients ( right ) ; black - mutations preserved between primary and PDX , blue - mutations , which were selected for / against in PDX models , green - relapse specific mutations selected for in INIPDX , absent or detected with a very low AF in primary patients ' diagnosis sample , orange - subclonal mutations lost in PDX , red - clonal mutations lost in PDX , grey - mutations , which AF was affected by low blast content ( BC ) at relapse Allele frequencies of all mutations preserved between primary patients ' samples ( x - axis ) and PDX samples ( y - axis ) ; PCC - Pearson ' s correlation

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY

Allele frequencies (AF) of mutations detected in primary (PRI) patients' samples (x-axis) at initial diagnosis (INI) and relapse (REL) plotted against AF of corresponding mutations detected in matched PDX models (y-axis) for relapse type 1 (left) and relapse type 2 patients (right); black - mutations preserved between primary and PDX, blue - mutations, which were selected for/against in PDX models, green - relapse specific mutations selected for in INIPDX, absent or detected with a very low AF in primary patients' diagnosis sample, orange - subclonal mutations lost in PDX, red - clonal mutations lost in PDX, grey - mutations, which AF was affected by low blast content (BC) at relapse Allele frequencies of all mutations preserved between primary patients' samples (x-axis) and PDX samples (y-axis); PCC- Pearson's correlation coefficient


Unsupervised hierarchical clustering based on the average degree of methylation of the 500 most variable promoters ( red - low / violet - high methylation levels ) ; * - relapse ( REL ) sample of patient P4 with blast content of 8 %

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Unsupervised hierarchical clustering based on the average degree of methylation of the 500 most variable promoters (red - low/violet -high methylation levels); * - relapse (REL) sample of patient P4 with blast content of 8%


The degree of the mean promoter methylation in patients ' samples plotted against the degree of mean promoter methylation in PDX samples ; ρ - Spearman ' s rank correlation coefficient ; median combined ranks across hexagonal bins are shown as a gradient according to the color

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The degree of the mean promoter methylation in patients' samples plotted against the degree of mean promoter methylation in PDX samples; ρ - Spearman's rank correlation coefficient; median combined ranks across hexagonal bins are shown as a gradient according to the color legend


TSS enrichment score of primary samples ( PRI ) and

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TSS enrichment score of primary samples (PRI) and PDXs


Reads number per peak in primary samples ( x - axis ) plotted against the reads number in corresponding PDX model ; PCC = Pearson ' s correlation coefficient )

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Reads number per peak in primary samples (x-axis) plotted against the reads number in corresponding PDX model; PCC=Pearson's correlation coefficient)


Fraction of peaks shared between PDX and corresponding primary leukemia for ATAC peaks with a certain height ( x - axis ; average number of reads that map into an accessible region called by Macs2 )

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Fraction of peaks shared between PDX and corresponding primary leukemia for ATAC peaks with a certain height (x-axis; average number of reads that map into an accessible region called by Macs2)


MA plot of log2 fold changes between primary and PDX samples plotted against the mean of normalized read counts per peak , red - differentially accessible regions ( p < 0 . 05 ) as analyzed by

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MA plot of log2 fold changes between primary and PDX samples plotted against the mean of normalized read counts per peak, red - differentially accessible regions (p<0.05) as analyzed by DESeq


Relative expression levels of the HIF1A - AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT - PCR analysis and represented as 39 minus Ct values .

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Relative expression levels of the HIF1A-AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT-PCR analysis and represented as 39 minus Ct values.


Sanger sequencing validation of RNA editing events on HIF1A - AS2 . The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1 . Asterisks mark the sites undergoing statistically significant increases in RNA editing .

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Sanger sequencing validation of RNA editing events on HIF1A-AS2. The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1. Asterisks mark the sites undergoing statistically significant increases in RNA editing.


RNA - IP of ADAR1 in HUVECs , followed by real - time PCR analysis of the HIF - 1A and HIF1A - AS2 RNAs . The relative enrichment of transcript association was normalized to the IgG . GAPDH was used as a non - binding control .

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RNA-IP of ADAR1 in HUVECs, followed by real-time PCR analysis of the HIF-1A and HIF1A-AS2 RNAs. The relative enrichment of transcript association was normalized to the IgG. GAPDH was used as a non-binding control.


Cells were cultured under normal or hypoxic condition . Relative levels of the HIF1A - AS transcripts in total vs . poly ( A ) + RNA fractions ( F ) were determined by qRT - PCR .

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Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F) were determined by qRT-PCR.


Cells were cultured under normal or hypoxic condition . Relative levels of the HIF1A - AS transcripts in subcellular fractions ( G ) , were determined by qRT - PCR .

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Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in subcellular fractions (G), were determined by qRT-PCR.


FISH analysis of the spatial distribution of HIF - 1A mRNA ( red ) and HIF1A - AS2 transcripts ( white ) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs . Nuclei were counterstained by DAPI . Scale bars , 10 μm .

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FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm.


Expression of HIF1A - AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides ( ODN ) - based knockdown . Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition , and subjected to gene expression analyses : The RNA abundance of HIF - 1A and HIF1A - AS2 was analyzed by real - time PCR and normalized by 18s rRNA ( A ) .

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O

Expression of HIF1A-AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides (ODN)-based knockdown. Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition, and subjected to gene expression analyses: The RNA abundance of HIF-1A and HIF1A-AS2 was analyzed by real-time PCR and normalized by 18s rRNA (A).


HIF - 1α protein level was monitored by western blot , with β - actin as the internal control ( B ) .

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HIF-1α protein level was monitored by western blot, with β-actin as the internal control (B).


Nuclear run - on assays were performed to assess the extent of nascent transcription of target genes under mock vs . CoCl2 treatment ( C )

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Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes under mock vs. CoCl2 treatment (C)


Nuclear run - on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A - AS2 knockdown cells ( D ) .

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Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A-AS2 knockdown cells (D).


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) and exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment ( E ) , The precipitated DNA fragments were quantitatively analyzed by real - time PCR using primers for the HIF1A - AS2 and HIF - 1A promoters , and normalized to the values of IgG . HIF1A - AS2 corresponds to the transcribed region .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment (E), The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) and exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A - AS2 - knockdown vs . control cells ( F ) . The precipitated DNA fragments were quantitatively analyzed by real - time PCR using primers for the HIF1A - AS2 and HIF - 1A promoters , and normalized to the values of IgG . HIF1A - AS2 corresponds to the transcribed region .

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A-AS2-knockdown vs. control cells (F). The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . The expression levels of ADAR1 , HIF - 1A and HIF1A - AS2 transcripts were analyzed by qRT - PCR .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The expression levels of ADAR1, HIF-1A and HIF1A-AS2 transcripts were analyzed by qRT-PCR.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Nuclear run - on assay was done to measure the newly synthesized HIF - 1A and HIF1A - AS2 RNAs in ADAR1 - knockdown cells , followed by real - time PCR analysis of the indicated transcripts .

O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Nuclear run-on assay was done to measure the newly synthesized HIF-1A and HIF1A-AS2 RNAs in ADAR1-knockdown cells, followed by real-time PCR analysis of the indicated transcripts.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . ChIP analysis of RNA polII binding on chromatin region of the HIF1A - AS2 and HIF - 1A promoters . The extent of occupancy was compared between control and ADAR1 - knockdown cells . The precipitated DNA fragments were quantitatively analyzed by real - time PCR and normalized by the values of IgG .

O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. ChIP analysis of RNA polII binding on chromatin region of the HIF1A-AS2 and HIF-1A promoters. The extent of occupancy was compared between control and ADAR1-knockdown cells. The precipitated DNA fragments were quantitatively analyzed by real-time PCR and normalized by the values of IgG.


Chromatin binding of ADAR1 to HIF1A - AS2 and HIF - 1A promoters under nomoxia ( 20 % O2 ) vs . hypoxia ( 1 % O2 , 5 hrs ) conditions , as assessed by ChIP .

B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O

Chromatin binding of ADAR1 to HIF1A-AS2 and HIF-1A promoters under nomoxia (20% O2) vs. hypoxia (1% O2, 5 hrs) conditions, as assessed by ChIP.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . The 3 ' - end extension of HIF1A - AS2 RNA transcription in ADAR1 knockdown cells ( GFP , control ) was examined by PCR with primers corresponding to the depicted regions of HIF1A - AS2 locus . GAPDH serves as an internal control . Sequence locations are shown in kbp and relative to the transcription start site .

O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The 3'-end extension of HIF1A-AS2 RNA transcription in ADAR1 knockdown cells (GFP, control) was examined by PCR with primers corresponding to the depicted regions of HIF1A-AS2 locus. GAPDH serves as an internal control. Sequence locations are shown in kbp and relative to the transcription start site.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α protein ( F ) by western

O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α protein (F) by western blot


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α downstream genes ( G ) by real - time PCR , as indicated . qRT - PCR results were normalized by the expression of 18s rRNA .

O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α downstream genes (G) by real-time PCR, as indicated. qRT-PCR results were normalized by the expression of 18s rRNA.


Transcriptome - wide changes in the cells subjected to CoCl2 treatment , HIF1A - AS2 or ADAR1 knockdown were profiled by RNA - Sequencing . For genes that undergo up - regulation upon CoCl2 treatment ( n = 505 ) , overall expression profiles of the different cell groups are represented by the heatmap . The expression values ( represented by normalized read counts ) were displayed in shades of red or blue ( linear scale ) relative to the means of all corresponding values within individual experimental groups . Clusters of genes ( based on unsupervised hierarchical clustering ) are indicated and denoted by their experimental types / conditions .

O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Transcriptome-wide changes in the cells subjected to CoCl2 treatment, HIF1A-AS2 or ADAR1 knockdown were profiled by RNA-Sequencing. For genes that undergo up-regulation upon CoCl2 treatment (n = 505), overall expression profiles of the different cell groups are represented by the heatmap. The expression values (represented by normalized read counts) were displayed in shades of red or blue (linear scale) relative to the means of all corresponding values within individual experimental groups. Clusters of genes (based on unsupervised hierarchical clustering) are indicated and denoted by their experimental types/conditions.


GSEA comparing the gene expression profile by gene up - regulated upon CoCl2 treatment and those down - regulated in ADAR1 knockdown . The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets . The bar ‐ code plot below enrichment score plot denote the rank of each member in the CoCl2 up - regulated gene list .

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GSEA comparing the gene expression profile by gene up-regulated upon CoCl2 treatment and those down-regulated in ADAR1 knockdown. The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets. The bar‐code plot below enrichment score plot denote the rank of each member in the CoCl2 up-regulated gene list.


HUVECs were transfected with the indicated combinations of siRNA ( GFP as control versus ADAR1 ) and ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . The expression levels of ADAR1 and HIF - 1α protein ( E ) were analyzed respectively by immunoblotting .

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HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. The expression levels of ADAR1 and HIF-1α protein (E) were analyzed respectively by immunoblotting.


HUVECs were transfected with the indicated combinations of siRNA ( GFP as control versus ADAR1 ) and ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . For cells in ( E ) , RNA expression of heat shock protein - encoding genes ( HSPA1A , HSPA1B , and HSPA6 ) with reported roles in angiogenesis were profiled by qRT - PCR .

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HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. For cells in (E), RNA expression of heat shock protein-encoding genes (HSPA1A, HSPA1B, and HSPA6) with reported roles in angiogenesis were profiled by qRT-PCR.


Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation . Scale bars , 2000 μm .

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Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation. Scale bars, 2000 μm.


Sanger sequencing validation of RNA editing of the LIMD1 ( A ) and VHL ( B ) transcripts .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O

Sanger sequencing validation of RNA editing of the LIMD1 (A) and VHL (B) transcripts.


Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs . Cells were then subjected to hypoxia treatment for 5 hrs . The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1 , VHL , PHD1 , PHD2 , ADAR1 and HIF - 1α , as indicated β - actin was used as loading control .

O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs. Cells were then subjected to hypoxia treatment for 5 hrs. The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1, VHL, PHD1, PHD2, ADAR1 and HIF-1α, as indicated β-actin was used as loading control.


To characterize subcellular distribution of target transcripts , real - time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O

To characterize subcellular distribution of target transcripts, real-time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments.


Translation efficiency was determined by quantifying transcript association with the polyribosome fraction , using ADAR1 - depleted MCF7 cells with hypoxia treatment . The abundance of LIMD1 in polysomes was analyzed by real - time PCR and normalized by the eEF1α levels .

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Translation efficiency was determined by quantifying transcript association with the polyribosome fraction, using ADAR1-depleted MCF7 cells with hypoxia treatment. The abundance of LIMD1 in polysomes was analyzed by real-time PCR and normalized by the eEF1α levels.


To inspect HIF - 1α protein abundance and modification with ubiquitin , anti - HIF - 1α , anti - ubiquitylation ( Ub ) , and anti - VHL immunoprecipitation experiments were performed , coupled with immunoblotting detection of ubiquitylation and HIF - 1α , as indicated . MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used . Protein signals were quantified using the ImageJ software . The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input ( In ) , with the control ( siGFP ) group being represented as 1 .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

To inspect HIF-1α protein abundance and modification with ubiquitin, anti- HIF-1α, anti-ubiquitylation (Ub), and anti-VHL immunoprecipitation experiments were performed, coupled with immunoblotting detection of ubiquitylation and HIF-1α, as indicated. MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used. Protein signals were quantified using the ImageJ software. The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input (In), with the control (siGFP) group being represented as 1.


B , Western blot showing no detectable Wbp2 protein in 4 week old mutant brain compared to wt littermate controls . 5μg of the protein lysate were subjected to 10 % SDS - PAGE . β - tubulin was used as loading control . Wbp2 l and Wbp2 s refer to the long and short isoforms respectively .

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B, Western blot showing no detectable Wbp2 protein in 4 week old mutant brain compared to wt littermate controls. 5μg of the protein lysate were subjected to 10% SDS-PAGE. β-tubulin was used as loading control. Wbp2 l and Wbp2 s refer to the long and short isoforms respectively.


C , Quantitative real - time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants ( n = 3 ) inner ears and eyes , compared to wt littermate controls ( n = 3 ) . Heterozygotes show intermediate levels . Hprt was used as a control and levels are normalised to wt levels .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

C, Quantitative real-time PCR showing severe knockdown of Wbp2 transcription in 4 week old mutants (n=3) inner ears and eyes, compared to wt littermate controls (n=3). Heterozygotes show intermediate levels. Hprt was used as a control and levels are normalised to wt levels.


D , F , X - gal staining of Wbp2 hets at P14 showing Wbp2 expression ( blue ) in all the main cochlear structures : the stria vascularis ( black arrowhead in D ) , spiral prominence ( empty arrowhead in D ) , Reissner ' s membrane ( arrow in D ) , strong expression in the spiral ganglion cells ( red arrowheads in E ) , IHCs and OHCs in the organ of Corti ( arrowheads in F ) , pillar cells and spiral limbus ( empty arrowhead in F ) . Scale bars : D : 50μm , E , F : 20μm . ihc : inner hair cells ; ohc : outer hair cells . No X - gal staining is observed in wt controls ( not shown ) . The X - gal reaction is always cytoplasmic .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

D, F, X-gal staining of Wbp2 hets at P14 showing Wbp2 expression (blue) in all the main cochlear structures: the stria vascularis (black arrowhead in D), spiral prominence (empty arrowhead in D), Reissner's membrane (arrow in D), strong expression in the spiral ganglion cells (red arrowheads in E), IHCs and OHCs in the organ of Corti (arrowheads in F), pillar cells and spiral limbus (empty arrowhead in F). Scale bars: D: 50μm, E,F: 20μm. ihc: inner hair cells; ohc: outer hair cells. No X-gal staining is observed in wt controls (not shown). The X-gal reaction is always cytoplasmic.


A , E Mean ABR thresholds ( ± SD ) for clicks and tone pips are plotted for wt ( green ) , het ( blue ) and hom ( red ) mice at ages A , P14 ( wt , n = 3 ; het , n = 8 ; hom , n = 6 ) ; B , 4 weeks ( wt , n = 38 ; het , n = 26 ; hom , n = 37 ) ; C , 14 weeks ( wt , n = 10 ; hom , n = 14 ) ; D , 28 weeks ( wt , n = 15 ; het , n = 5 ; hom , n = 25 ) ; E , 44 weeks ( wt , n = 9 ; het , n = 2 ; hom , n = 11 ) . Grey symbols and lines indicate thresholds of individual mutants . In B , open symbols represent thresholds under urethane anaesthesia ( see methods ) , showing no difference compared with ketamine / xylazine used for all other thresholds . F , Mean thresholds for mutants aged 2 weeks ( yellow ) , 4 weeks ( purple ) , 14 weeks ( cyan ) , 28 weeks ( grey ) and 44 weeks ( black ) .

O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,E Mean ABR thresholds (± SD) for clicks and tone pips are plotted for wt (green), het (blue) and hom (red) mice at ages A, P14 (wt, n=3; het, n=8; hom, n=6); B, 4 weeks (wt, n=38; het, n=26; hom, n=37); C, 14 weeks (wt, n=10; hom, n=14); D, 28 weeks (wt, n=15; het, n=5; hom, n=25); E, 44 weeks (wt, n=9; het, n=2; hom, n=11). Grey symbols and lines indicate thresholds of individual mutants. In B, open symbols represent thresholds under urethane anaesthesia (see methods), showing no difference compared with ketamine/xylazine used for all other thresholds.F, Mean thresholds for mutants aged 2 weeks (yellow), 4 weeks (purple), 14 weeks (cyan), 28 weeks (grey) and 44 weeks (black).


G , Averaged click - evoked ABR waveforms from 4 weeks old wt ( n = 23 , green ) and mutants ( n = 34 , red ) , at 50dB sensation level ( SL ) ( left panel ) . SP and ABR Wave 1 ( W1 ) are indicated by grey areas . Expanded averaged SP and ABR W1 waveform patterns for 10 - 60dB SL in 10dB increments are plotted in green ( wt ; middle panel ) and red ( mutants ; right panel ) . , to illustrate the growth of SP and W1 with stimulus level .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, Averaged click-evoked ABR waveforms from 4 weeks old wt (n=23, green) and mutants (n=34, red), at 50dB sensation level (SL) (left panel). SP and ABR Wave 1 (W1) are indicated by grey areas. Expanded averaged SP and ABR W1 waveform patterns for 10-60dB SL in 10dB increments are plotted in green (wt; middle panel) and red (mutants; right panel)., to illustrate the growth of SP and W1 with stimulus level.


H , I . Mean 2f1 - f2 DPOAE thresholds ( ± SD ) are plotted for wt ( green ) , heterozygote ( blue ) and homozygous ( red ) mice aged 4 weeks ( H ) or 21 weeks ( I ) , as a function of f2 frequency .

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H, I. Mean 2f1-f2 DPOAE thresholds (± SD) are plotted for wt (green), heterozygote (blue) and homozygous (red) mice aged 4 weeks (H) or 21 weeks (I), as a function of f2 frequency.


A . Pure tone audiogram recorded from one of the probands , showing severe to profound bilateral asymmetric hearing loss .

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A. Pure tone audiogram recorded from one of the probands, showing severe to profound bilateral asymmetric hearing loss.


B . Agarose gel trace of cDNA obtained from mouse wt P28 inner ear ( IE ) , P28 brain , adult and P4 organ of Corti ( OC ) . Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28 . If we look at just the organ of Corti ( adult and P4 ) , we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform , which was not even picked up by sequencing ( see C ) . Wbp2 l : long isoform ( 550 bp ) ; Wbp2 s : short isoform ( 480 bp ) .

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B. Agarose gel trace of cDNA obtained from mouse wt P28 inner ear (IE), P28 brain, adult and P4 organ of Corti (OC). Results show expression of two Wbp2 isoforms in the brain at P28 and very faint band for the short isoform together with a strong band for the long isoform in the inner ear at P28. If we look at just the organ of Corti (adult and P4), we observe a strong band for the long isoform in the P4 OC and an almost undetectable band for the short isoform, which was not even picked up by sequencing (see C). Wbp2 l: long isoform (550 bp); Wbp2 s: short isoform (480 bp).


D . Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28 , with a short trace of the short isoform showing up only when a higher concentration of protein lysate ( 20μg ) is loaded . Both isoform are absent in the Wbp2 - deficient mouse . Gapdh was used as a loading control . Wbp2 l : long isoform ; Wbp2 s : short isoform .

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D. Western blot showing the predominant presence of the Wbp2 long isoform in the cochlea at P28, with a short trace of the short isoform showing up only when a higher concentration of protein lysate (20μg) is loaded. Both isoform are absent in the Wbp2-deficient mouse. Gapdh was used as a loading control. Wbp2 l: long isoform; Wbp2 s: short isoform.


A . At P14 , afferent terminals below IHCs are slightly swollen in the mutants ( neurofilament labelling in green , arrowheads to compare ; CtBP2 labels ribbons and IHCnuclei in red ) . Scale bars : 10 μm . At P28 , Neurofilament / CtBP2 labelling in the organ of Corti of 4 week old Wbp2 - deficient mice and littermate controls shows severe swelling of IHCafferent terminals in the mutants , especially in the 24kHz region ( yellow arrowheads ) . The pre - synaptic ribbons do not look as well aligned to the terminals in the mutants ( white arrows ) . At this stage , we also observe swelling of OHCafferent terminals in the 24kHz region ( empty arrowheads ) . Scale bars : 5 μm . ihc : IHCnucleus ; p : pillar side ; m : modiolar side . B . Counts of pre - synaptic ribbons per IHC in the 8 , 18 and 24kHz regions , showing no difference between mutants and controls at P28 .

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A. At P14, afferent terminals below IHCs are slightly swollen in the mutants (neurofilament labelling in green, arrowheads to compare; CtBP2 labels ribbons and IHCnuclei in red). Scale bars: 10 μm. At P28,Neurofilament/CtBP2 labelling in the organ of Corti of 4 week old Wbp2-deficient mice and littermate controls shows severe swelling of IHCafferent terminals in the mutants, especially in the 24kHz region (yellow arrowheads). The pre-synaptic ribbons do not look as well aligned to the terminals in the mutants (white arrows). At this stage, we also observe swelling of OHCafferent terminals in the 24kHz region (empty arrowheads). Scale bars: 5 μm. ihc: IHCnucleus; p: pillar side; m: modiolar side.B. Counts of pre-synaptic ribbons per IHC in the 8, 18 and 24kHz regions, showing no difference between mutants and controls at P28.


C . TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells ( arrowheads for comparisons between mutants and controls ) . Scale bars : 5 μm .

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C. TEM of the organ of Corti performed at P28 showing swollen afferent terminals below inner and outer hair cells (arrowheads for comparisons between mutants and controls). Scale bars: 5 μm.


D . Neurofilament / CtBP2 labelling shows swollen and retracting terminals ( white arrows ) , especially in the 24kHz regions in the mutants at 8 weeks ( arrows ) . Scale bars : 10μm . E . Counts of pre - synaptic ribbons in the 8 , 18 and 24kHz regions , showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks .

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D. Neurofilament/CtBP2 labelling shows swollen and retracting terminals (white arrows), especially in the 24kHz regions in the mutants at 8 weeks (arrows). Scale bars: 10μm.E. Counts of pre-synaptic ribbons in the 8, 18 and 24kHz regions, showing no difference in their number per IHC in the mutants compared to littermate controls at 8 weeks.


A . We arrayed synapse images aligned by ascending size of the post - synaptic site after GluR2 / 3 and CtBP2 labelling . This is a composite image made of several synapses taken from a single IHC from a single wt and a single hom , representing double labelling experiments performed on 3 mutants and 3 controls . Synapses in the mutants show abnormal morphology and smaller green patches , suggesting reduced expression of the GluR2 / 3 AMPA receptor subunits . Scale bar at bottom right : 1μm .

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A. We arrayed synapse images aligned by ascending size of the post-synaptic site after GluR2/3 and CtBP2 labelling. This is a composite image made of several synapses taken from a single IHC from a single wt and a single hom, representing double labelling experiments performed on 3 mutants and 3 controls. Synapses in the mutants show abnormal morphology and smaller green patches, suggesting reduced expression of the GluR2/3 AMPA receptor subunits. Scale bar at bottom right: 1μm.


B - G . TEM images of IHC ribbon synapses of wt ( B , arrowheads for synaptic vesicles ) and homs ( C - G ) at 4 weeks of age , showing a representative array of synaptic phenotypes in the 24 kHz cochlear region of mutants . While in C and D the ribbons look slightly abnormal in size with misplaced synaptic vesicles ( arrowheads in C ) , we also observe orphan post - synaptic densities surrounded by floating synaptic vesicles ( arrowheads in E ) , ribbons with synaptic membranes ( arrow in F ) that have detached from the IHC membrane ( the asterisk labels the original position of the synapse before detachment ) and are floating in the swollen nerve terminal ( F ) , ribbons ( arrow in G ) that are detached from their densities ( asterisk in G ) . Scale bars : B - E : 200 nm ; F , G : 500 nm . nt : nerve terminal ; psd : post - synaptic density ; snt : swollen nerve terminal .

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B-G. TEM images of IHC ribbon synapses of wt (B, arrowheads for synaptic vesicles) and homs (C-G) at 4 weeks of age, showing a representative array of synaptic phenotypes in the 24 kHz cochlear region of mutants. While in C and D the ribbons look slightly abnormal in size with misplaced synaptic vesicles (arrowheads in C), we also observe orphan post-synaptic densities surrounded by floating synaptic vesicles (arrowheads in E), ribbons with synaptic membranes (arrow in F) that have detached from the IHC membrane (the asterisk labels the original position of the synapse before detachment) and are floating in the swollen nerve terminal (F), ribbons (arrow in G) that are detached from their densities (asterisk in G). Scale bars: B-E: 200 nm; F,G: 500 nm. nt: nerve terminal; psd: post-synaptic density; snt: swollen nerve terminal.


B , Quantitative real - time PCR showing reduced mRNA levels for Esr1 , Esr2 and Pgr and upregulation of Shank3 and Psd - 95 in cochleae of 4 week old Wbp2 - deficient mice compared to littermate controls ( n = 3 for each genotype ) . Hprt is used as relative control . Error bars : ± SD . Two - tailed student t test : p = 0 . 03 for Psd - 95 ; p = 0 . 007 for Shank3 ; p = 0 . 03 for Esr2 ; p = 0 . 0016 for Esr1 ; p = 0 . 037 for Pgr .

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B, Quantitative real-time PCR showing reduced mRNA levels for Esr1, Esr2 and Pgr and upregulation of Shank3 and Psd-95 in cochleae of 4 week old Wbp2-deficient mice compared to littermate controls (n=3 for each genotype). Hprt is used as relative control. Error bars: ±SD. Two-tailed student t test: p=0.03 for Psd-95; p=0.007 for Shank3; p=0.03 for Esr2; p=0.0016 for Esr1; p=0.037 for Pgr.


C , Synapses from one mutant and one control IHC at 4 weeks of age after Psd - 95 and CtBP2 labelling , showing stronger Psd - 95 expression in the mutants compared to controls , representing double labelling experiments performed on 3 mutants and 3 controls . P indicates direction of pillar cells . Scale bar : 10μm . D , Quantification of Psd95 fluorescence in IHC synapses , representing expression in the apical ( 9kHz best frequency region of the cochlea ) and basal ( 24kHz best frequency region of the cochlea ) regions at 4 weeks old . Data from 2 wt and 2 homs were analysed ( 16 synapses per cochlear region per mouse ) . AU , Arbitrary Units . Wt : 24kHz 22 . 41 ± 10 . 70 , 9kHz 11 . 045 ± 2 . 128 ; Mutants : 24kHz 66 . 73 ± 13 . 70 , p = 0 . 069 ; 9kHz : 26 . 02 ± 5 . 79 p = 0 . 075 .

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C, Synapses from one mutant and one control IHC at 4 weeks of age after Psd-95 and CtBP2 labelling, showing stronger Psd-95 expression in the mutants compared to controls, representing double labelling experiments performed on 3 mutants and 3 controls. P indicates direction of pillar cells. Scale bar: 10μm.D, Quantification of Psd95 fluorescence in IHC synapses, representing expression in the apical (9kHz best frequency region of the cochlea) and basal (24kHz best frequency region of the cochlea) regions at 4 weeks old . Data from 2 wt and 2 homs were analysed (16 synapses per cochlear region per mouse). AU, Arbitrary Units.Wt: 24kHz 22.41±10.70, 9kHz 11.045±2.128; Mutants: 24kHz 66.73±13.70, p=0.069; 9kHz: 26.02±5.79 p=0.075.


( A ) Representative micrographs of optic nerve sections collected from mice 8 days after ONC , showing the accumulation of IB - 4 + activated myeloid cells in the injury site , which is demarcated by GFAP staining . Scale bar , 50 µm .

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(A) Representative micrographs of optic nerve sections collected from mice 8 days after ONC, showing the accumulation of IB-4+ activated myeloid cells in the injury site, which is demarcated by GFAP staining. Scale bar, 50 µm.


( B ) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [ Cx3cr1gfp / + WT ] BM chimeric mice , immunostained for GFP ( red ) , to depict monocyte - derived macrophages , and IB - 4 ( green ) to label activated myeloid cells . Scale bar , 100 µm . Inset scale , 20 µm .

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(B) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [Cx3cr1gfp/+ WT] BM chimeric mice, immunostained for GFP (red), to depict monocyte-derived macrophages, and IB-4 (green) to label activated myeloid cells. Scale bar, 100 µm. Inset scale, 20 µm.


( C ) Representative micrograph of optic nerve section 7 days after crush injury , immunostained for CD3 ( red , arrows ) . Scale bar , 100 µm .

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(C) Representative micrograph of optic nerve section 7 days after crush injury, immunostained for CD3 (red, arrows). Scale bar, 100 µm.


( D ) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina . Leukocytes were identified by gating on CD45 + cells , and were further gated on CD11b + or TCRβ + to detect monocytes / macrophages and T cells , respectively . Of the CD11b + cells , infiltrating monocytes were regarded as CD45hi .

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(D) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina. Leukocytes were identified by gating on CD45+ cells, and were further gated on CD11b+ or TCRβ+ to detect monocytes/macrophages and T cells, respectively. Of the CD11b+ cells, infiltrating monocytes were regarded as CD45hi.


( E ) Representative contour plot from [ Cx3cr1gfp / + WT ] BM chimeric mouse , verifying that infiltrating blood - derived macrophages ( CX3CR1 - GFP + ) are indeed CD45hi , and showing that they are detectable only in retinas from eyes that underwent ONC , while absent in noninjured controls ( red vs . blue line ) . Cells were pre - gated on CD11b . n = 3 - 4 per group .

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(E) Representative contour plot from [Cx3cr1gfp/+ WT] BM chimeric mouse, verifying that infiltrating blood-derived macrophages (CX3CR1-GFP+) are indeed CD45hi, and showing that they are detectable only in retinas from eyes that underwent ONC, while absent in noninjured controls (red vs. blue line). Cells were pre-gated on CD11b. n = 3-4 per group.


( F ) Quantitative analysis of flow cytometry data , showing the induction of an immune response in the retina after ONC , evident by a general increase in leukocytes ( CD45 + ) , including CD11b + CD45hi cells ( representing infiltrating macrophages ) and T cells ( TCRβ + ) . n = 4 per group . Data shown are representative of six independent experiments . Graphs show mean ± SE of each group . * * * P < 0 . 001 . FSC - forward scatter ; noninj - noninjured control ; ONC - optic nerve crush .

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(F) Quantitative analysis of flow cytometry data, showing the induction of an immune response in the retina after ONC, evident by a general increase in leukocytes (CD45+), including CD11b+CD45hi cells (representing infiltrating macrophages) and T cells (TCRβ+). n = 4 per group. Data shown are representative of six independent experiments. Graphs show mean ± SE of each group. *** P < 0.001. FSC - forward scatter; noninj - noninjured control; ONC - optic nerve crush.


( A , C - E ) Changes in mRNA transcript levels of an array of adhesion molecules ( A ) in retinas from ONC eyes compared with contralateral noninjured retinas , 8 hours , and 1 , 3 and 7 days after ONC . n = 6 - 13 per group . Data shown are representative of three independent experiments .

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(A, C-E) Changes in mRNA transcript levels of an array of adhesion molecules (A) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC. n = 6-13 per group. Data shown are representative of three independent experiments.


( B ) Representative micrographs showing the expression of ICAM - 1 and VCAM - 1 ( red ) by the RPE ( green ) 12 hours after ONC . Scale bar , 20 µm . Bar graphs throughout the figure show mean ± SE of each group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different time points are indicated by asterisks between bars . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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(B) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 12 hours after ONC. Scale bar, 20 µm. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


( A , C - E ) Changes in mRNA transcript levels of an array of chemokines ( C , D ) and cytokine receptors ( E ) in retinas from ONC eyes compared with contralateral noninjured retinas , 8 hours , and 1 , 3 and 7 days after ONC ( optic nerve crush ) . n = 6 - 13 per group . Data shown are representative of three independent experiments .

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(A, C-E) Changes in mRNA transcript levels of an array of chemokines (C, D) and cytokine receptors (E) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC (optic nerve crush). n = 6-13 per group. Data shown are representative of three independent experiments.


( A , B ) Quantitative flow cytometry analysis of myeloid cells in the RPE complex ( A ) and retinas ( B ) from injured ( filled bars ) and contralateral noninjured eyes ( empty bars ) , on days 1 , 3 and 7 after ONC . Cells were pre - gated on total leukocytes ( CD45 + ) , and analyzed for the expression of CD11b . CD11b + cells were further discriminated based on their expression levels of Ly6C . n = 6 - 7 per group . Bar graphs throughout the figure show mean ± SE of each group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different time points are indicated by asterisks between bars . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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(A, B) Quantitative flow cytometry analysis of myeloid cells in the RPE complex (A) and retinas (B) from injured (filled bars) and contralateral noninjured eyes (empty bars), on days 1, 3 and 7 after ONC. Cells were pre-gated on total leukocytes (CD45+), and analyzed for the expression of CD11b. CD11b+ cells were further discriminated based on their expression levels of Ly6C. n = 6-7 per group. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


Mice were subjected to ONC and i . v . - injected with either an anti - VCAM - 1 blocking antibody or an isotype control antibody . Eyes were collected after 2 or 3 days for flow cytometry . ( A ) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti - VCAM - 1 antibody , as compared to isotype - treated controls . n = 3 per group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different antibody treatments are indicated by asterisks between bars .

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Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for flow cytometry. (A) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti-VCAM-1 antibody, as compared to isotype-treated controls. n = 3 per group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different antibody treatments are indicated by asterisks between bars.