Figure 6Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice ( A ) and their density in the respective regions ( B ) . 3 animals were analyzed for each genotype . The number of quantified cilia is indicated ( n ) . Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor ( Tubacin ) and appropriate controls ( Nitubacin and DMSO , C ) Colonies were stained with crystal violetClonogenicity analysis of CLESCs treated with siArl13b and its siControl . Colonies were stained with crystal violetsiRNA knockdown efficiency of Arl13b ( E ) in Ki67 FUCCI - CLESCs was evaluated real - time RT - PCR analysis . The results are in arbitrary values after normalization for GapDHsiRNA knockdown efficiency of Hdac6 ( F ) in Ki67 FUCCI - CLESCs was evaluated real - time RT - PCR analysis . The results are in arbitrary values after normalization for GapDHFlow cytometry analysis and quantification of samples indicated in ( E and F ) . Representative IF double staining and 3D reconstruction of the Prom1 ( green ) and AcTub ( red ) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant ( top panels ) . Black - white inserts show Prom1 signals only . Same cells were analyzed by SEM ( bottom panels ) . Note that Prom1 is asymmetrically distributed along the primary cilium ( white arrows ) as observed in vivo ( see Fig 2C ) and mutation K138Q significantly impacts on the primary cilium length . MV , microvilli . IF staining and 3D reconstruction of AcTub - labeled ( red ) primary cilium at the apical membrane of parental MDCK cells ( MDCK ) and those cells transfected with Prom1 or its K138Q mutant . The images were used to quantify the primary cilium length ( Appendix Figs S6B and C ) . Arrows indicate primary cilia with more than 4 μm in length . Co - immunoisolation ( IS ) analyses were performed with paramagnetic beads using antibodies against either Arl13b ( K ) Recovered materials were analyzed by immunoblotting against the indicated proteins . Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum - associated forms of Prom1 , respectively , while arrows show Arl13b . Arrowhead , immunoglobulin . Co - immunoisolation ( IS ) analyses were performed with paramagnetic beads using antibodies against either Prom1 ( AC133 ) or with anti - FITC antibody as control ( L ) . Recovered materials were analyzed by immunoblotting against the indicated proteins . Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum - associated forms of Prom1 , respectively , while arrows show Arl13b . Arrowhead , immunoglobulin . The ratio Arl13b / Prom1 immunoreactivities was quantified ( n = 3 ) ( M ) .

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Figure 6Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice (A) and their density in the respective regions (B). 3 animals were analyzed for each genotype. The number of quantified cilia is indicated (n).Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor (Tubacin) and appropriate controls (Nitubacin and DMSO, C) Colonies were stained with crystal violetClonogenicity analysis of CLESCs treated with siArl13b and its siControl. Colonies were stained with crystal violetsiRNA knockdown efficiency of Arl13b (E) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDHsiRNA knockdown efficiency of Hdac6 (F) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDHFlow cytometry analysis and quantification of samples indicated in (E and F).Representative IF double staining and 3D reconstruction of the Prom1 (green) and AcTub (red) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant (top panels). Black-white inserts show Prom1 signals only. Same cells were analyzed by SEM (bottom panels). Note that Prom1 is asymmetrically distributed along the primary cilium (white arrows) as observed in vivo (see Fig 2C) and mutation K138Q significantly impacts on the primary cilium length. MV, microvilli.IF staining and 3D reconstruction of AcTub-labeled (red) primary cilium at the apical membrane of parental MDCK cells (MDCK) and those cells transfected with Prom1 or its K138Q mutant. The images were used to quantify the primary cilium length (Appendix Figs S6B and C). Arrows indicate primary cilia with more than 4 μm in length.Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Arl13b (K) Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Prom1 (AC133) or with anti-FITC antibody as control (L). Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.The ratio Arl13b/Prom1 immunoreactivities was quantified (n = 3) (M).


Figure 7Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by phase - contrast microscopy ( A ) Box - whisker plots show the number of domes per 600 mm2 ( A ) . Three images were analyzed per experiment ( n = 4 ) . 4X magnification ( inset in panel A ) . Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM ( B , top panels ) or confocal laser scanning microscopy after DAPI staining ( B , bottom panels , Individual cells within a given dome are indicated ( B , top panels ) and were quantified using three different planes ( bottom , centre and top ) of DAPI - labeled nuclei ( B , bottom panels ) . Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc ( A - C ) or as indicated ( D ) . confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated ( C ) . Means and standard deviations are displayed . 15 domes per independent experiment were examined ( n = 3 ) . Cell density at a given time of culture ( 7 , 10 and 14 days ) was determined by counting DAPI - labeled nuclei per 0 . 1 mm2 ( D , n = 4 ) . Cilia with a length of > 4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM ( arrows ) . Mutation in the Hdac6 - binding site of Prom1 increases the release of Prom1 + vesicles . 16 h - conditioned medium was subjected to differential centrifugation for 5 min at 300 x g ( P1 ) ; 20 min at 1 , 200 x g ( P2 ) ; 30 min at 10 , 000 x g ( P3 ) and 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent cell lysates were prepared . The entire pellets and 1 / 40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1 , AcTub and β - actin ( F ) . Molecular mass markers are indicated . All three proteins were detected in the vesicle fractions ( P3 and P4 ) suggesting Prom1 + vesicles are released from primary cilia and microvilli . Mutation in the Hdac6 - binding site of Prom1 increases the release of Prom1 + vesicles . 16 h - conditioned medium was subjected to differential centrifugation for 5 min at 300 x g ( P1 ) ; 20 min at 1 , 200 x g ( P2 ) ; 30 min at 10 , 000 x g ( P3 ) and 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent cell lysates were prepared . The release of Prom1 + vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions ( G )

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Figure 7Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by phase-contrast microscopy (A) Box-whisker plots show the number of domes per 600 mm2 (A). Three images were analyzed per experiment (n = 4). 4X magnification (inset in panel A).Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM (B, top panels) or confocal laser scanning microscopy after DAPI staining (B, bottom panels, Individual cells within a given dome are indicated (B, top panels) and were quantified using three different planes (bottom, centre and top) of DAPI-labeled nuclei (B, bottom panels).Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc (A-C) or as indicated (D). confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated (C). Means and standard deviations are displayed. 15 domes per independent experiment were examined (n = 3). Cell density at a given time of culture (7, 10 and 14 days) was determined by counting DAPI-labeled nuclei per 0.1 mm2 (D, n = 4).Cilia with a length of >4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM (arrows).Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The entire pellets and 1/40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1, AcTub and β-actin (F). Molecular mass markers are indicated. All three proteins were detected in the vesicle fractions (P3 and P4) suggesting Prom1+ vesicles are released from primary cilia and microvilli.Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The release of Prom1+ vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions (G)


Figure 8The mRNA expression of SHH effector genes ( Gli1 / 2 and Glis1 - 3 ) on laser microdissection captured samples were evaluated by real - time RT - PCR analysis . The results are in arbitrary values after normalization for GapDH . Each sample was isolated from distinct mice ( n = 3 ) . Representative IF staining of Gli1 ( left panel , red ) and Glis2 ( right panel , red ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Dotted lines , basement membrane . 3D reconstruction of Glis2 staining ( see B , red ) and its association with DAPI - labeled nuclei ( blue ) . Nuclear transparency was set at 0 or 70 % as indicated . Representative IF double staining of Glis2 ( red , bottom panel ) and AcTub ( green , top and bottom panels ) along stem cell - transit amplifying cell axis in lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions . Volume of Sox2 - positive region on the lower incisor CLE at P7 was evaluated in WT vs . Glis2 KO mice upon IF staining . Number of animals analyzed is indicated ( n ) . Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop ( top panels , green ) or cytoplasmic C - terminal end ( bottom panels , red ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice . Samples are counterstained with DAPI ( blue ) . Dotted lines , basement membrane . Arrowheads marks approximate boundary of stem cell regions . Representative IF double staining of Glis2 ( red ) and AcTub ( green ) as a marker of primary cilia in stem cell region of WT vs . Prom1 KO mice . Note the reduction of Glis2 expression in Prom1 - deficient animals .

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Figure 8The mRNA expression of SHH effector genes (Gli1/2 and Glis1-3) on laser microdissection captured samples were evaluated by real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH. Each sample was isolated from distinct mice (n = 3).Representative IF staining of Gli1 (left panel, red) and Glis2 (right panel, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Dotted lines, basement membrane.3D reconstruction of Glis2 staining (see B, red) and its association with DAPI-labeled nuclei (blue). Nuclear transparency was set at 0 or 70% as indicated.Representative IF double staining of Glis2 (red, bottom panel) and AcTub (green, top and bottom panels) along stem cell-transit amplifying cell axis in lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions.Volume of Sox2-positive region on the lower incisor CLE at P7 was evaluated in WT vs. Glis2 KO mice upon IF staining. Number of animals analyzed is indicated (n).Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop (top panels, green) or cytoplasmic C-terminal end (bottom panels, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice. Samples are counterstained with DAPI (blue). Dotted lines, basement membrane. Arrowheads marks approximate boundary of stem cell regions.Representative IF double staining of Glis2 (red) and AcTub (green) as a marker of primary cilia in stem cell region of WT vs. Prom1 KO mice. Note the reduction of Glis2 expression in Prom1-deficient animals.


Figure 9Proximity ligation assay analysis of Prom1 and Glis2 association ( red ) in stem cell and transit amplifying cell regions of lower incisor CLE at P7 . As negative control , one conjugated primary antibody ( anti - Glis2 ) was omitted . SCs , stem cells ; TACs , transit amplifying cells . Time dependent Prom1 and Glis2 co - expression ( proximity ligation assay ) in the cultured CLESCs starved for 4 days , and then treated with SHH ( 100 ng / ml ) and importazole ( 40 µM ) , an inhibitor of importin β1function . DMSO was used as vehicle control . Data are represented triplicated samples as mean and standard deviation . PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . Arrows indicated genes with expression changed for more than 2 folder increase or decrease . PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . The mRNA expression of gene significantly changed in the PCR array ( in ( C ) ) ( D ) was evaluated in WT , Prom1 and Glis2 KO mice by real - time RT - PCR analysis . Each sample was isolated from distinct mice ( n = 3 ) . The results are in arbitrary values after normalization for GapDH . PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . The mRNA expression of gene significantly changed in the Stat pathway members ( E ) was evaluated in WT , Prom1 and Glis2 KO mice by real - time RT - PCR analysis . Each sample was isolated from distinct mice ( n = 3 ) . The results are in arbitrary values after normalization for GapDH . Chromatin IP analysis of binding of Glis2 to Stat3 promoter ( see Appendix Fig S9D ) in the absence or presence of SHH at different concentration as indicated on triplicated samples , using specific anti - Glis2 antibodies and rabbit IgG as control .

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Figure 9Proximity ligation assay analysis of Prom1 and Glis2 association (red) in stem cell and transit amplifying cell regions of lower incisor CLE at P7. As negative control, one conjugated primary antibody (anti-Glis2) was omitted. SCs, stem cells; TACs, transit amplifying cells.Time dependent Prom1 and Glis2 co-expression (proximity ligation assay) in the cultured CLESCs starved for 4 days, and then treated with SHH (100 ng/ml) and importazole (40 µM), an inhibitor of importin β1function. DMSO was used as vehicle control. Data are represented triplicated samples as mean and standard deviation.PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). Arrows indicated genes with expression changed for more than 2 folder increase or decrease.PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). The mRNA expression of gene significantly changed in the PCR array (in (C)) (D) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH.PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). The mRNA expression of gene significantly changed in the Stat pathway members (E) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH.Chromatin IP analysis of binding of Glis2 to Stat3 promoter (see Appendix Fig S9D) in the absence or presence of SHH at different concentration as indicated on triplicated samples, using specific anti-Glis2 antibodies and rabbit IgG as control.


Figure 3A ) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks ( GW ) , 10 GW , and 12 GW and in the adult human thyroid : quantitative PCR results normalized to one of three thyroid tissues at 8 GW value ( in black ) and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus 8 GW ( in black ) using ANOVA test showed no significant differencesB ) Tubb1 is expressed in the developing mouse thyroid at E13 . 5 , E15 . 5 , and E17 . 5 and in the adult mouse thyroid : quantitative PCR results normalized to one of three thyroid tissues at E13 . 5 value and peptidylprolyl isomerase A . Experiments with at least three tissues per stage . Statistical comparisons versus E13 . 5 ( black ) , * * p < 0 . 01 using ANOVA testC ) Sorted mouse thyroid cells , Tubb1 expression in endothelial cells ( Pecam - positive cells ) , epithelial cells ( Epcam ) , fibroblasts ( Pdgfra ) , and leucocytes ( CD45 ) at E17 . 5 and adulthood : quantitative PCR results ( normalized to Epcam - positive cells at adult stage and peptidylprolyl isomerase A ) . Mean ± SEM of three independent cell - sorting experiments . Statistical comparisons versus epithelial cells ( in black ) at each stage , * p < 0 . 05 using ANOVA testD ) Immunohistofluorescence of β1 - tubulin ( in red ) , thyroglobulin ( TG , in green ) , and both merged ( from left to right ) at 12 GW in human thyroid tissue . The boxes delineate the enlarged areas shown belowF ) Localization of transfected Myc - tagged wild - type and mutant ( P160L ) β1 - tubulin in Nthy cells . Immunostaining with anti - Myc antibody ( in green ) and microtubule cytoskeleton stained with α - tubulin antibody ( in red ) . Note that β1 - tubulin - P160L is found throughout the cytoplasm showing puncta appearance , whereas wild - type β1 - tubulin colocalizes with the

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Figure 3A) TUBB1 is expressed in the developing human thyroid at 8 gestational weeks (GW), 10 GW, and 12 GW and in the adult human thyroid: quantitative PCR results normalized to one of three thyroid tissues at 8 GW value (in black) and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus 8 GW (in black) using ANOVA test showed no significant differencesB) Tubb1 is expressed in the developing mouse thyroid at E13.5, E15.5, and E17.5 and in the adult mouse thyroid: quantitative PCR results normalized to one of three thyroid tissues at E13.5 value and peptidylprolyl isomerase A. Experiments with at least three tissues per stage. Statistical comparisons versus E13.5 (black), **p<0.01 using ANOVA testC) Sorted mouse thyroid cells, Tubb1 expression in endothelial cells (Pecam-positive cells), epithelial cells (Epcam), fibroblasts (Pdgfra), and leucocytes (CD45) at E17.5 and adulthood: quantitative PCR results (normalized to Epcam-positive cells at adult stage and peptidylprolyl isomerase A). Mean±SEM of three independent cell-sorting experiments. Statistical comparisons versus epithelial cells (in black) at each stage, *p<0.05 using ANOVA testD) Immunohistofluorescence of β1-tubulin (in red), thyroglobulin (TG, in green), and both merged (from left to right) at 12 GW in human thyroid tissue. The boxes delineate the enlarged areas shown belowF) Localization of transfected Myc-tagged wild-type and mutant (P160L) β1-tubulin in Nthy cells. Immunostaining with anti-Myc antibody (in green) and microtubule cytoskeleton stained with α-tubulin antibody (in red). Note that β1-tubulin -P160L is found throughout the cytoplasm showing puncta appearance, whereas wild-type β1-tubulin colocalizes with the microtubules


Figure 4A ) Thyroid morphology was investigated using Nkx2 - 1 staining at E9 . 5 and E11 . 5 ( sagittal sections ) and at E13 . 5 , E15 . 5 , and E17 . 5 ( transverse sections ) in wild - type ( wt ) and Tubb1 - / - littermates . Nkx2 - 1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes . Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11 . 5 and E13 . 5 ( arrow ) , respectively , in Tubb1 - / - mice . At E11 . 5 , stained migrating thyroid cells were visible along the tract only in Tubb1 - / - littermates ( arrow ) . At E17 . 5 , a pyramidal lobe was visible in Tubb1 - / - littermates ( arrow ) . tr : trachea ; ub : ultimobranchial body . Scale bar : 50 μB ) Total thyroid surface area ( μm2 ) was quantified using Nkx2 - 1 staining at each embryonic stage . Top : thyroid surface area normalised for weight of each embryo . Bottom : thyroid surface area in μm2 . Right : proliferation ratio calculated as the proportion of Nkx2 - 1 - positive cells labelled with Ki67 at E9 . 5 in wt and Tubb1 - / - thyroid anlages . ma : median anlage . Note that the thyroid ma is larger and the proliferation ratio higher at E . 9 . 5 in Tubb1 - / - mice , whereas from E13 . 5 onwards the Tubb1 - / - thyroids were hypoplastic . Wt in black , KO , Tubb1 - / - in greC ) Thyroid marker expression by quantitative PCR in thyroid tissue at E15 . 5 and E17 . 5 in Tubb1 - / - versus wt mice normalised for peptidylprolyl isomerase A . Note the decreases in thyroid differentiation markers at E15 . 5 and E17 . 5 and also in thyroid transcription factors at E17 . 5D ) Staining of Nkx2 - 1 and T4 at E17 . 5 and surface area quantification demonstrating T4 retention in Tubb1 - / - versus wt thyroids . The data are the percentage of T4 or calcitonin ( CT ) surface area versus total thyroid area ( estimated from the Nkx2 - 1 - stained surface area ) . Scale bar : 50 μm . Student " s test , * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . Experiments were done on at least three tissues per stage for each

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Thyroid morphology was investigated using Nkx2-1 staining at E9.5 and E11.5 (sagittal sections) and at E13.5, E15.5, and E17.5 (transverse sections) in wild-type (wt) and Tubb1-/- littermates. Nkx2-1was used as a marker of early thyrocyte progenitors and differentiated thyrocytes. Delays in thyroid migration and in fusion of the median anlage to the ultimobranchial bodies were observed at E11.5 and E13.5 (arrow), respectively, in Tubb1-/- mice. At E11.5, stained migrating thyroid cells were visible along the tract only in Tubb1-/- littermates (arrow). At E17.5, a pyramidal lobe was visible in Tubb1-/- littermates (arrow). tr: trachea; ub: ultimobranchial body. Scale bar: 50 μB) Total thyroid surface area (μm2) was quantified using Nkx2-1 staining at each embryonic stage. Top: thyroid surface area normalised for weight of each embryo. Bottom: thyroid surface area in μm2. Right: proliferation ratio calculated as the proportion of Nkx2-1-positive cells labelled with Ki67 at E9.5 in wt and Tubb1-/- thyroid anlages. ma: median anlage. Note that the thyroid ma is larger and the proliferation ratio higher at E.9.5 in Tubb1-/- mice, whereas from E13.5 onwards the Tubb1-/- thyroids were hypoplastic. Wt in black, KO, Tubb1-/- in greC) Thyroid marker expression by quantitative PCR in thyroid tissue at E15.5 and E17.5 in Tubb1-/- versus wt mice normalised for peptidylprolyl isomerase A. Note the decreases in thyroid differentiation markers at E15.5 and E17.5 and also in thyroid transcription factors at E17.5D) Staining of Nkx2-1 and T4 at E17.5 and surface area quantification demonstrating T4 retention in Tubb1-/- versus wt thyroids. The data are the percentage of T4 or calcitonin (CT) surface area versus total thyroid area (estimated from the Nkx2-1-stained surface area). Scale bar: 50 μm. Student"s test, *p<0.05, **p<0.01 and ***p<0.001. Experiments were done on at least three tissues per stage for each gen


Figure 5A ) Serum TSH and T4 levels in 3 - month - old Tubb1 - / - and wild - type mice . Numbers of animals tested were 10 wild - type males and 14 Tubb1 - / - males for TSH and for 14 wild - type and 22 Tubb1 - / - males for T4 . Tubb1 - / - mice had hypothyroidism with elevated TSH and decreased T4 versus wild - type mice . Student " s test , * * p < B ) Nkx2 - 1 ( in brown ) immunostaining of adult thyroid tissue . Note the disorganisation of the thyroid tissue in Tubb1 - / - versus wild - type miceC ) Ultrastructural alterations shown by electron microscopy in wild - type and Tubb1 - / - thyroid tissues . In Tubb1 - / - tissues , note the disorganisation of secretion vesicles ( white asterisks ) and rods of identical density to secretion vesicles ( white arrow ) . The ER is considerably dilated in Tubb1 - / - thyrocytes . Representative views . Scale bars at the bottom left for each view . Co : colloiD ) ER stress in Tubb1 - / - thyroid tissue . Top : Chop and XBPs expression by quantitative PCR in adults , normalized to peptidylprolyl isomerase A . Experiments with at least three tissues per stage for each genotype ; wt in black and Tubb1 - / - in grey . Bottom : Chop and Actin protein expression by western blotting in three representative wild - type and Tubb1 - / - mice . The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1 - / - versus wild - type thyroids . All lanes are from the same blot , which was cut where indicated . Student " s test , * p < 0 . 05 and * * * p < E ) Co - immunostaining of endoplasmic reticulum ( ER ) marker ( KDEL , in red ) , thyroglobulin ( Tg in green ) , and both merged ( from left to right ) in adult thyroid tissue . Note the thyrocyte disorganisation in Tubb1 - / -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Serum TSH and T4 levels in 3-month-old Tubb1-/- and wild-type mice. Numbers of animals tested were 10 wild-type males and 14 Tubb1-/- males for TSH and for 14 wild-type and 22 Tubb1-/-males for T4. Tubb1-/- mice had hypothyroidism with elevated TSH and decreased T4 versus wild-type mice. Student"s test, ** p<B) Nkx2-1 (in brown) immunostaining of adult thyroid tissue. Note the disorganisation of the thyroid tissue in Tubb1-/- versus wild-type miceC) Ultrastructural alterations shown by electron microscopy in wild-type and Tubb1-/- thyroid tissues. In Tubb1-/- tissues, note the disorganisation of secretion vesicles (white asterisks) and rods of identical density to secretion vesicles (white arrow). The ER is considerably dilated in Tubb1-/- thyrocytes. Representative views. Scale bars at the bottom left for each view. Co: colloiD) ER stress in Tubb1-/- thyroid tissue. Top: Chop and XBPs expression by quantitative PCR in adults, normalized to peptidylprolyl isomerase A. Experiments with at least three tissues per stage for each genotype; wt in black and Tubb1-/- in grey. Bottom: Chop and Actin protein expression by western blotting in three representative wild-type and Tubb1-/- mice. The Chop band quantification normalised for Actin confirms the increased Chop expression demonstrated by quantitative PCR in Tubb1-/- versus wild-type thyroids. All lanes are from the same blot, which was cut where indicated. Student"s test, *p<0.05 and ***p<E) Co-immunostaining of endoplasmic reticulum (ER) marker (KDEL, in red), thyroglobulin (Tg in green), and both merged (from left to right) in adult thyroid tissue. Note the thyrocyte disorganisation in Tubb1-/- mice


Figure 6A ) Representative blood smears of patients with TUBB1 mutations ( P1 and P3 homozygous for p . P160L , P4 heterozygous for p . Y106X , and P6 and P7 heterozygous for c . 35delG ) after May - Grünwald Giemsa staining . Variable platelet size in patients was observed with all three mutations with presence of macroplatelets ( arrows ) B ) Transmission electron microscopy ( TEM ) analysis of platelet ultrastructure in the patients . The results confirm the abnormal platelet morphologyC ) Representative images of proplatelet formation in P1 and P4 . Cultured control and patient megakaryocytes ( MKs ) after thrombopoietin - induced differentiation were spread over a BSA - coated coverslip on day 10 . On day 13 or 14 , MKs were fixed ( 4 % paraformaldehyde ) and proplatelet structure was observed after β - tubulin staining . Scale bars : 20 μm . Only P1 and P4 could be studied . The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions ( left ) and the diameter of coiled elements ( right ) of the future proplatelets , both of which were significantly increased in the patients , indicating that the TUBB1 mutations ( homozygous p . P160L and heterozygous p . Y106X ) affected proplatelet morphology . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * * p < 0 . 001 ; 20 - 60 MKs were analysed /

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Representative blood smears of patients with TUBB1 mutations (P1 and P3 homozygous for p.P160L, P4 heterozygous for p.Y106X, and P6 and P7 heterozygous for c.35delG) after May-Grünwald Giemsa staining. Variable platelet size in patients was observed with all three mutations with presence of macroplatelets (arrows)B) Transmission electron microscopy (TEM) analysis of platelet ultrastructure in the patients. The results confirm the abnormal platelet morphologyC) Representative images of proplatelet formation in P1 and P4. Cultured control and patient megakaryocytes (MKs) after thrombopoietin-induced differentiation were spread over a BSA-coated coverslip on day 10. On day 13 or 14, MKs were fixed (4% paraformaldehyde) and proplatelet structure was observed after β-tubulin staining. Scale bars: 20 μm. Only P1 and P4 could be studied. The graphs show the mean ± SEM of the shaft thickness of proplatelet extensions (left) and the diameter of coiled elements (right) of the future proplatelets, both of which were significantly increased in the patients, indicating that the TUBB1 mutations (homozygous p.P160L and heterozygous p.Y106X) affected proplatelet morphology. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, ***p<0.001; 20-60 MKs were analysed/pat


Figure 7A ) Relative expression of α - tubulin and β1 - tubulin in platelets from P1 and P3 ( homozygous p . P160L ) , P4 ( heterozygous p . Y106X ) , and P6 and P7 ( heterozygous c . 35delG ) was quantified by Western blotting using specific antibodies against α - tubulin or β1 - tubulin . With both mutations , α - tubulin expression was normal but β1 - tubulin expression was significantly decreased . The β1 - tubulin antibody recognizes C - terminal domainB ) αIIbβ3 integrin activation was evaluated by flow cytometry ( % of positive platelets and mean fluorescence intensity ) in whole blood from P1 and P3 ( homozygous p . P160L ) and from P6 and P7 ( heterozygous c . 35delG ) ; P4 could not be studied . While no αIIbβ3 integrin activations were detected in P6 and P7 ( family 3 ) , P1 and P3 ( family 1 ) exhibited a significant increase of αIIbβ3 activation , indicating basal platelet activationC ) Aggregation of washed platelets induced by ADP ( 5 or 10 μM ) or collagen ( 0 . 6 , 0 . 8 or 2 μg / mL ) was evaluated in at least one member of each family . Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls , suggesting that TUBB1 affected by homozygous p . P160L and heterozygous p . Y106X mutations affected platelet function . Platelet aggregation to both agonists was normal in both patients from family 3 ( heterozygous c . 35delG ) . Family F1 , n = 3 independent experiments ; Family F2 , n = 1 experiment ; Family F3 , n = 2 independent experiments . Statistical significance was determined by one - way ANOVA , followed by Dunnett " s multiple comparisons test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , nd : statistical significance not determined

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Relative expression of α-tubulin and β1-tubulin in platelets from P1 and P3 (homozygous p.P160L), P4 (heterozygous p.Y106X), and P6 and P7 (heterozygous c.35delG) was quantified by Western blotting using specific antibodies against α-tubulin or β1-tubulin. With both mutations, α-tubulin expression was normal but β1-tubulin expression was significantly decreased. The β1-tubulin antibody recognizes C-terminal domainB) αIIbβ3 integrin activation was evaluated by flow cytometry (% of positive platelets and mean fluorescence intensity) in whole blood from P1 and P3 (homozygous p.P160L) and from P6 and P7 (heterozygous c.35delG); P4 could not be studied. While no αIIbβ3 integrin activations were detected in P6 and P7 (family 3), P1 and P3 (family 1) exhibited a significant increase of αIIbβ3 activation, indicating basal platelet activationC) Aggregation of washed platelets induced by ADP (5 or 10 μM) or collagen (0.6, 0.8 or 2 μg/mL) was evaluated in at least one member of each family. Patients from families 1 and 2 had increased platelet aggregation in response to low doses of agonist versus controls, suggesting that TUBB1 affected by homozygous p.P160L and heterozygous p.Y106X mutations affected platelet function. Platelet aggregation to both agonists was normal in both patients from family 3 (heterozygous c.35delG). Family F1, n=3 independent experiments; Family F2, n=1 experiment; Family F3, n=2 independent experiments. Statistical significance was determined by one-way ANOVA, followed by Dunnett"s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, nd: statistical significance not determined because


Figure 1 ( A ) Srcap expression in murine different tissues was examined by Northern blot . A 277 nt probe of Srcap ( 81 - 358nt ) was labeled for Northern blot analysis . 18S RNA was used as a loading control . ( B ) Two - week - old SRCAP - C - HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining . A global look of the section was shown . Scale bar : 500 μm . Green : EpCAM , Red : HA tag , Nuclei were counterstained by DAPI . ( C ) Murine intestine tissues were fixed and stained according to iDISCO staining protocol . SRCAP was highly expressed in vast majority of crypts in mouse intestine . Scale bar : 50 μm . Green : EpCAM , Red : SRCAP , Nuclei were counterstained by DAPI . ( D ) Duodenum region of intestinal tissues were obtained from Lgr5GFP - CreERT2 ( Lgr5GFP ) mice for immunofluorescence staining by indicated antibodies with OpalTM 7 - color fIHC kit . Nuclei were counterstained with DAPI . Scale bar : 50 μm . ( E ) ISCs from Lgr5GFP mice were collected for organoid formation . Organoids were fixed for immunofluorescence staining with indicated antibodies . Scale bar : 50 μm . Data information : Green : EpCAM , Red : SRCAP , Purple : PCNA , Nuclei were counterstained by DAPI .

O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Srcap expression in murine different tissues was examined by Northern blot. A 277 nt probe of Srcap (81-358nt) was labeled for Northern blot analysis. 18S RNA was used as a loading control.(B) Two-week-old SRCAP-C-HA tag mice were sacrificed for longitudinal sections followed by immunofluorescence staining. A global look of the section was shown. Scale bar: 500 μm. Green: EpCAM, Red: HA tag, Nuclei were counterstained by DAPI.(C) Murine intestine tissues were fixed and stained according to iDISCO staining protocol. SRCAP was highly expressed in vast majority of crypts in mouse intestine. Scale bar: 50 μm. Green: EpCAM, Red: SRCAP, Nuclei were counterstained by DAPI.(D) Duodenum region of intestinal tissues were obtained from Lgr5GFP-CreERT2 (Lgr5GFP) mice for immunofluorescence staining by indicated antibodies with OpalTM 7-color fIHC kit. Nuclei were counterstained with DAPI. Scale bar: 50 μm.(E) ISCs from Lgr5GFP mice were collected for organoid formation. Organoids were fixed for immunofluorescence staining with indicated antibodies. Scale bar: 50 μm. Data information: Green: EpCAM, Red: SRCAP, Purple: PCNA, Nuclei were counterstained by DAPI.


Figure 2 ( A ) Representative images of intestines from 8 - week - old Srcap + / + and Srcap - / - mice ( n = 6 per group ) . Length of 120 villi and 120 crypts ( 20 villi and 20 crypts per mouse ) was measured and shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 100 μm . Dotted lines indicate the boundary between crypts and villi . Arrows indicate the length of crypts or villi . ( B ) Intestinal tissues from 8 - week - old Srcap + / + and Srcap - / - mice ( n = 6 per group ) were collected for immunofluorescence staining with indicated antibodies . Typical images were shown in left panel and statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 50 μm . ( C ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test . ( D ) Srcap + / + and Srcap - / - mice were treated with 10 Gy ' s radiation and sacrificed at different time points . Intestinal tissues were collected for H & E staining . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 100 μm . ( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - YFP ( LRYFP ) were crossed with Srcapflox / flox mice , followed by administration of tamoxifen ( TAM ) for lineage tracing analysis . Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained . Numbers of traced crypt - villus units were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . Scale bars , 50 μm . ( F ) Proliferation of Srcap + / + and Srcap - / - ISCs were examined by Ki67 staining followed by flow cytometry analysis . Percentages of proliferated cells were counted as means ± S . D ( lower panel ) . * * P < 0 . 01 by two - tailed Student ' s t - test . n = 5 per group . ( G ) Multiple color staining was performed in Srcap + / + and Srcap - / - intestine sections . AP - Red , DAB and AP - Blue were used for immunohistochemistray coloration . Numbers of each kind of epithelial cells from 50 crypts or 50 villi ( 10 crypts and 10 villi per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bars , 50 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative images of intestines from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) was measured and shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.(B) Intestinal tissues from 8-week-old Srcap+/+ and Srcap-/- mice (n=6 per group) were collected for immunofluorescence staining with indicated antibodies. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.(C) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.(D) Srcap+/+ and Srcap-/- mice were treated with 10 Gy's radiation and sacrificed at different time points. Intestinal tissues were collected for H&E staining. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 100 μm.(E) Lgr5GFP-CreERT2;Rosa26lsl-YFP (LRYFP) were crossed with Srcapflox/flox mice, followed by administration of tamoxifen (TAM) for lineage tracing analysis. Mice were sacrificed 7 days after TAM induction and typical jejunum sections were stained. Numbers of traced crypt-villus units were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5). Scale bars, 50 μm.(F) Proliferation of Srcap+/+ and Srcap-/- ISCs were examined by Ki67 staining followed by flow cytometry analysis. Percentages of proliferated cells were counted as means ± S.D (lower panel). ** P < 0.01 by two-tailed Student's t-test. n=5 per group.(G) Multiple color staining was performed in Srcap+/+ and Srcap-/- intestine sections. AP-Red, DAB and AP-Blue were used for immunohistochemistray coloration. Numbers of each kind of epithelial cells from 50 crypts or 50 villi (10 crypts and 10 villi per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bars, 50 μm.


Figure 3 ( A ) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti - SRCAP antibody or rabbit IgG followed by ADASDS - PAGE , sliver staining and mass spectrometry . ( B ) SRCAP and REST were stained in murine intestinal crypts . Scale bar , 10 μm . ( D ) Flag - tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti - Flag antibody . REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting . ( E ) C - terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay . ( F ) Intestinal crypt lysates were incubated with anti - REST antibody , followed by immunoblotting with indicated antibodies . ( G ) Intestinal crypt lysates were incubated with anti - REST antibody and protein A / G beads in 4 ℃ overnight . Recombinant SRCAP protein was added with different concentrations , followed by immunoblotting .

O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Nuclear extracts of murine intestinal crypts were immunoprecipitated with anti-SRCAP antibody or rabbit IgG followed by ADASDS-PAGE, sliver staining and mass spectrometry.(B) SRCAP and REST were stained in murine intestinal crypts. Scale bar, 10 μm.(D) Flag-tagged REST fragments were expressed in 293T and purified by immunoprecipitation with anti-Flag antibody. REST proteins were incubated with crypt lysates for pulldown followed by immunoblotting.(E) C-terminal domain of REST was sufficient and essential for SRCAP association by pulldown assay.(F) Intestinal crypt lysates were incubated with anti-REST antibody, followed by immunoblotting with indicated antibodies.(G) Intestinal crypt lysates were incubated with anti-REST antibody and protein A/G beads in 4℃ overnight. Recombinant SRCAP protein was added with different concentrations, followed by immunoblotting.


Figure 4 ( B ) Transcriptome microarray analysis between Srcap - / - ISCs and WT ISCs . Top ten downregulated TFs in Srcap - / - ISCs were listed . ( C ) Prdm16 expression was tested in Srcap + / + and Srcap - / - ISCs by Western blotting . ( D ) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining . Green : GFP ; Red : SRCAP ; Blue : Prdm16 . Scale bar , 10 μm . ( E ) Prdm16 promoter ( - 875 ~ - 854 ) region was used as probe to perform DNA - pulldown assay with murine intestinal crypt lysates followed by immunoblotting . ( F , G ) Indicated promoter activation was analyzed with luciferase assay . DKK1 was used as a control . Results of relative fold changes are shown as means ± S . D . Data represent five independent replicates . . * * p < 0 . 01 by two - tailed Student ' s t - test . . ( H ) 1 × 104 ISCs from indicated mice were collected for organoid formation . For gene depletion or restoration , ISCs were infected with lentivirus . Scale bar , 200 μm . Data information : All data represent five independent experiments .

O O B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Transcriptome microarray analysis between Srcap-/- ISCs and WT ISCs. Top ten downregulated TFs in Srcap-/- ISCs were listed.(C) Prdm16 expression was tested in Srcap+/+ and Srcap-/- ISCs by Western blotting.(D) Prdm16 expression was visualized in indicated intestinal tissues by immunofluorescence staining. Green: GFP; Red: SRCAP; Blue: Prdm16. Scale bar, 10 μm.(E) Prdm16 promoter (-875~-854) region was used as probe to perform DNA-pulldown assay with murine intestinal crypt lysates followed by immunoblotting.(F, G) Indicated promoter activation was analyzed with luciferase assay. DKK1 was used as a control. Results of relative fold changes are shown as means ± S.D. Data represent five independent replicates. . ** p<0.01 by two-tailed Student's t-test. .(H) 1×104 ISCs from indicated mice were collected for organoid formation. For gene depletion or restoration, ISCs were infected with lentivirus. Scale bar, 200 μm. Data information: All data represent five independent experiments.


Figure 5 ( A ) WT and Srcap - / - ISCs were conducted for transcriptome microarray , followed by Gene Set Enrichment Analysis ( GSEA ) analysis for PPARδ target genes . ( B ) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . ( C ) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining . Scale bar , 50 μm . ( D ) ISCs were collected for organoid formation followed by quantitative RT - PCR . For Prdm16 overexpression , ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . * * p < 0 . 01 by two - tailed Student ' s t - test . ( E ) mRNA levels of ISC signature genes were examined by quantitative RT - PCR . Primers were listed in Table EV1 . Relative gene expression folds were normalized to endogenous β - actin and shown as means ± S . D . Data represent five independent replicates . ( F ) Representative images of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test . ( lower panel ) . n = 6 for each group . GW501516 ( 10 μM ) were added directly into organoid culture medium .

O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) WT and Srcap-/- ISCs were conducted for transcriptome microarray, followed by Gene Set Enrichment Analysis (GSEA) analysis for PPARδ target genes.(B) Expression levels of PPARs and PPAR target genes were assessed in sorted ISCs by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 and *** p<0.001 by two-tailed Student's t-test.(C) PPARδ expression was visualized in intestinal tissues by immunofluorescence staining. Scale bar, 50 μm.(D) ISCs were collected for organoid formation followed by quantitative RT-PCR. For Prdm16 overexpression, ISCs were infected with lentivirus carrying Prdm16 overexpressing plasmids. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates. ** p<0.01 by two-tailed Student's t-test.(E) mRNA levels of ISC signature genes were examined by quantitative RT-PCR. Primers were listed in Table EV1. Relative gene expression folds were normalized to endogenous β-actin and shown as means ± S.D. Data represent five independent replicates.(F) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test. (lower panel). n=6 for each group. GW501516 (10 μM) were added directly into organoid culture medium.


Figure 6 ( A ) Representative images of intestines from 8 - week - old Rest + / + and Rest - / - mice ( n = 6 per group ) . Length of 120 villi and 120 crypts ( 20 villi and 20 crypts per mouse ) were measured in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 100 μm . Dotted lines indicate the boundary between crypts and villi . Arrows indicate the length of crypts or villi . ( B ) 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 3 for each group . Organoid formation ratios was shown as means ± S . D . * * p < 0 . 01 by two - tailed Student ' s t - test . ( C ) Intestine tissues from 8 - week - old Rest + / + and Rest - / - mice ( n = 6 per group ) were collected for immunohistochemistry staining . Typical images were shown in left panel and statistical ISC numbers from 120 crypts ( 20 crypts per mouse ) were shown in right panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . Scale bar , 50 μm . ( D ) Immunofluorescence staining of intestine tissues from Srcapfl / fl ; Lgr5GFP - CreERT2 and Restfl / fl ; Lgr5GFP - CreERT2 mice . Scale bar , 50 μm . ( E ) Lgr5GFP - CreERT2 ; Rosa26lsl - lacZ ( LRlacZ ) were crossed with Rest - / - mice , followed by TAM administration for lineage tracing for intestinal whole - mount staining for β - gal ( upper panel ) . Mice were sacrificed after one week and typical sections were shown ( middle panel ) . Scale bar , 200 μm . Numbers of traced crypt - villus units were calculated as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . 50 fields ( 10 fields per mouse ) were calculated for each group ( n = 5 ) ( lower panel ) . ( F ) Rest + / + and Rest - / - mice were treated with 10 Gy ' s radiation and sacrificed at the indicated time points . Intestinal tissues were collected for H & E staining . Scale bars , 100 μm . Numbers of intact crypts were shown in lower panel as means ± S . D . * * P < 0 . 01 by two - tailed Student ' s t - test . 50 fields ( 10 fields per mouse ) were taken for each group ( n = 5 ) . ( G ) Representative images of organoid formation from indicated ISCs were shown ( upper panel ) . Scale bar , 200 μm . n = 6 for each group . Organoid number per well was counted as means ± S . D . ( lower panel ) . Lentivirus - carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM - treated LRCas9 mice to obtain Prdm16 - / - and Ppard - / - ISCs . 1 × 104 ISCs from indicated mice were collected for organoid formation . Scale bar , 200 μm . n = 5 for each group .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Representative images of intestines from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group). Length of 120 villi and 120 crypts (20 villi and 20 crypts per mouse) were measured in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 100 μm. Dotted lines indicate the boundary between crypts and villi. Arrows indicate the length of crypts or villi.(B) 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=3 for each group. Organoid formation ratios was shown as means ± S.D. ** p<0.01 by two-tailed Student's t-test.(C) Intestine tissues from 8-week-old Rest+/+ and Rest-/- mice (n=6 per group) were collected for immunohistochemistry staining. Typical images were shown in left panel and statistical ISC numbers from 120 crypts (20 crypts per mouse) were shown in right panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. Scale bar, 50 μm.(D) Immunofluorescence staining of intestine tissues from Srcapfl/fl; Lgr5GFP-CreERT2 and Restfl/fl; Lgr5GFP-CreERT2 mice. Scale bar, 50 μm.(E) Lgr5GFP-CreERT2;Rosa26lsl-lacZ (LRlacZ) were crossed with Rest-/- mice, followed by TAM administration for lineage tracing for intestinal whole-mount staining for β-gal (upper panel) . Mice were sacrificed after one week and typical sections were shown (middle panel). Scale bar, 200 μm. Numbers of traced crypt-villus units were calculated as means± S.D. ** P < 0.01 by two-tailed Student's t-test in right panel. 50 fields (10 fields per mouse) were calculated for each group (n=5) (lower panel).(F) Rest+/+ and Rest-/- mice were treated with 10 Gy's radiation and sacrificed at the indicated time points. Intestinal tissues were collected for H&E staining. Scale bars, 100 μm. Numbers of intact crypts were shown in lower panel as means± S.D. ** P < 0.01 by two-tailed Student's t-test. 50 fields (10 fields per mouse) were taken for each group (n=5).(G) Representative images of organoid formation from indicated ISCs were shown (upper panel). Scale bar, 200 μm. n=6 for each group. Organoid number per well was counted as means ± S.D. (lower panel). Lentivirus-carried sgPrdm16 and sgPpard were prepared in 293T cells and infected into ISCs derived from TAM-treated LRCas9 mice to obtain Prdm16-/- and Ppard-/- ISCs. 1×104 ISCs from indicated mice were collected for organoid formation. Scale bar, 200 μm. n=5 for each group.


Figure 1A , B Quantification of mitotic ( A ) and meiotic ( B ) cells in the distal germline in wild - type animals fed bacteria transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) Data information : Data are presented as mean ± SE ( A ) N = 3 at least 5 worms per replicate and condition , ( B ) N = 3 at least 10 worms for conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsQuantification of dissected distal germline from wild - type animal stained either anti - phosphohistone H3 ( anti - PH3 ) ( C relative to the total number of mitotic cells of wild - type animal Data informatio Data are presented a as mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsQuantification of dissected distal germline from wild - type animal stained eithe anti - CDK - 1 ( anti - CDK - 1 ( D relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsQuantification of dissected distal germline from wild - type animal stained eithe 5 - ethynyl - 2 ′ - deoxyuridine ( EdU - green ) ( E ) relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsF Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild - type worms fed as in ( A ) Data information : Data are presented as mean ± SE ( F ) N = 5 at least 10 worms per replicate and condition . * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B Quantification of mitotic (A) and meiotic (B) cells in the distal germline in wild-type animals fed bacteria transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) Data information: Data are presented as mean ± SE (A) N=3 at least 5 worms per replicate and condition, (B) N=3 at least 10 worms for conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsQuantification of dissected distal germline from wild-type animal stained either anti-phosphohistone H3 (anti-PH3) (C relative to the total number of mitotic cells of wild-type animal Data informatio Data are presented a as mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsQuantification of dissected distal germline from wild-type animal stained eithe anti-CDK-1 (anti-CDK-1 (D relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsQuantification of dissected distal germline from wild-type animal stained eithe 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsF Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild-type worms fed as in (A) Data information: Data are presented as mean ± SE (F) N=5 at least 10 worms per replicate and condition. * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Figure 2Quantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiation 125 Gy ( A Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit ) , UVC 400J / m2 ( B Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment o wild - type strain relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit different doses of UVB ( C Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit 100µM cisplatin Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiation 125 Gy (A Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit ), UVC 400J/m2 (B Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment o wild-type strain relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit different doses of UVB (C Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit 100µM cisplatin Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Figure 3Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 ( A and left untreated or exposed to 1200 J / m2 UVB . Apoptotic cells were counted 12 , 24 and 36 hours after exposure Data information : Data are presented as mean ± SEM , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsTime course quantification of apoptotic corpses relative to the total number of meitic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) o isp - 1 ( B ) , and left untreated or exposed to 1200 J / m2 UVB . Apoptotic cells were counted 12 , 24 and 36 hours after exposure Data information : Data are presented as mean ± SEM , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOV Tukey " s multiple comparisonsReal Time PCR gene expression analysis of egl - genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiatio Data information : Data are presented a mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsReal Time PCR gene expression analysis of egl - genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit UV Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsReal Time PCR gene expression analysis o ced - 1 genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiatio Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsReal Time PCR gene expression analysis o ced - 1 genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treate with UV Data information : Data are presented as mean ± SE N = F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s mu comparisons

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 (A and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsTime course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) o isp-1 (B), and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOV Tukey"s multiple comparisonsReal Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented a mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsReal Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit UV Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsReal Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsReal Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treate with UV Data information: Data are presented as mean ± SE N= F). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s mu comparisons test


Figure 4Representative fluorescence pictures and quantification ( below the fluorescence pictures ) of dissected adult gonads relative to the number of mitotic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 and stained with DAPI ( blue ) and either anti - PARP - 1 ( red ) ( A Scale bar : 20µ ( A Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and conditionRepresentative fluorescence pictures and quantification ( below the fluorescence pictures ) of dissected adult gonads relative to the number of mitotic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 and stained with DAPI ( blue and eithe anti - RPA - 1 ( green ) and anti - RAD - 51 ( red ) antibodies and treated with gamma radiation ( 125 Gy ) ( B ) ( untreated animals displayed no staining of RPA - 1 and RAD - 51 , not shown ) 15µm ( B Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and conditionQuantification of mitotic cells in wild - type animals fed as ( A ) and left untreated or treated with gamma radiation 125 G Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and conditionQuantification of mitotic cells in wild - type animals fed as ( A ) and left untreated or treate with 1200 J / m2 UV Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A Scale bar: 20µ (A Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and conditionRepresentative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue and eithe anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown) 15µm (B Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and conditionQuantification of mitotic cells in wild-type animals fed as (A) and left untreated or treated with gamma radiation 125 G Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and conditionQuantification of mitotic cells in wild-type animals fed as (A) and left untreated or treate with 1200 J/m2 UV Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition