Electron micrographs showing ultrastructures of cotyledon cells of germinatedV . mungo seeds ( A - G ) , immunogold localization of α - amylase in cotyledon cells ( H ) , and ultrastructure of cells of detached cotyledons ( I ) . Toluidine blue ( TB ) staining of sections from cotyledons of day 3 V . mungo seedlings . Conversion from TB - stained cells ( region I ) to TB - stainless cells ( region III ) was accompanied with that of the PSV to LV . ( B ) SGs and PSVs in TB - stained cells ( A , region I ) . The PSV was filled with storage proteins . Arrowheads indicate border between SGs and the cytoplasm . ( C ) SGs and PSVs in cotyledons at region II in A . Electron density of PSVs became low . SGs were surrounded with membranous structure ( arrows ) . LED areas were found between SGs and the cytoplasm . ( D ) SG and LV in TB - stainless cells ( region III in A ) . The PSV was converted to the LV in the cells . The areas around SG with LED were enlarged . ( E ) Ultrastructure of TB - stainless cells . SGs wrapped with a LED area contacted with LVs ( arrowheads ) . Vesicles with similar density to LVs were observed . ( F ) LED membranes around the SGs fused with the LV membranes ( arrow ) . ( G ) SGs were observed in LVs . The shape of SGs were largely different from those in B - E . ( H ) An Immunogold image representing degradation of SGs by α - amylase localized in LVs . Gold particles from anti - α - amylase antibody were densely detected in the peripheral region of SGs , which is inserted into the inside of the LV . ( I ) PSVs were not converted to LVs , and SGs were not taken up into PSVs in the cells of detached cotyledons . Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells . LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars : ( A ) 50 μm ; ( E and G ) 2 μm ; ( D , F , and I ) 1 μm ; ( B , C , and H ) 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A-G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B-E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti-α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.


LysoTracker red staining of partially broken cotyledon cells , SGs , or cotyledon sections . Cotyledon cells , SGs , and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods . ( A ) Differential interface - contrast image of a broken cell . ( B ) Fluorescent image of A . ( C ) Differential interface - contrast image of a broken cell . ( D ) Fluorescent image of C . ( E ) Differential interface - contrast image of cells of a section from attached cotyledons . ( F ) Fluorescent image of E . ( G ) Magnified image of E . ( H ) Magnified image of F . ( I ) Differential interface - contrast image of an SG . ( J ) Fluorescent image of I . ( K ) Differential interface - contrast image of an SG . ( L ) Fluorescent image of K . ( M ) Differential interface - contrast image of cells of a section from detached cotyledons . ( N ) Fluorescent image of M . ( O ) Magnified image of M . ( P ) Magnified image of O . Bars : ( A - D , G - L , O , and P ) 20 μm ; ( E , F , M , and N ) 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LysoTracker red staining of partially broken cotyledon cells, SGs, or cotyledon sections. Cotyledon cells, SGs, and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods. (A) Differential interface-contrast image of a broken cell. (B) Fluorescent image of A. (C) Differential interface-contrast image of a broken cell. (D) Fluorescent image of C. (E) Differential interface-contrast image of cells of a section from attached cotyledons. (F) Fluorescent image of E. (G) Magnified image of E. (H) Magnified image of F. (I) Differential interface-contrast image of an SG. (J) Fluorescent image of I. (K) Differential interface-contrast image of an SG. (L) Fluorescent image of K. (M) Differential interface-contrast image of cells of a section from detached cotyledons. (N) Fluorescent image of M. (O) Magnified image of M. (P) Magnified image of O. Bars: (A-D, G-L, O, and P) 20 μm; (E, F, M, and N) 50 μm.


Electron micrographs showing autophagosome - mediated autophagy in attached ( A - C ) and detached ( D and E ) cotyledon cells . ( A ) Two autophagosomes containing cytoplasm were observed . Mitochondria and the cytoplasm appeared to be enclosed with an ER - like double membrane . ( B ) An autophagosome containing mitochondria and cytoplasm was fused with LV ( arrowheads ) . ( C ) Autophagic body containing mitochondria was found in LV . ( D ) An autophagosome containing mitochondria and cytoplasm was observed . The PSV was not converted into the LV . ( E ) Autophagic body was observed in the PSV of detached cotyledon cells . AB , autophagic body ; AP , autophagosome ; ER , endoplasmic reticulum ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars , 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron micrographs showing autophagosome-mediated autophagy in attached (A-C) and detached (D and E) cotyledon cells. (A) Two autophagosomes containing cytoplasm were observed. Mitochondria and the cytoplasm appeared to be enclosed with an ER-like double membrane. (B) An autophagosome containing mitochondria and cytoplasm was fused with LV (arrowheads). (C) Autophagic body containing mitochondria was found in LV. (D) An autophagosome containing mitochondria and cytoplasm was observed. The PSV was not converted into the LV. (E) Autophagic body was observed in the PSV of detached cotyledon cells. AB, autophagic body; AP, autophagosome; ER, endoplasmic reticulum; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


( B , C ) HEK293T cells were co ‐ transfected with the indicated constructs . Cell lysates were subjected to IP using anti ‐ FLAG antibodies . The resulting precipitates were examined by immunoblot analysis with anti ‐ FLAG and anti ‐ HA antibodies .

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(B,C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were subjected to IP using anti‐FLAG antibodies. The resulting precipitates were examined by immunoblot analysis with anti‐FLAG and anti‐HA antibodies.


( D ) HEK293T cells were cultured in regular or starvation medium for 2 h . Cells were harvested and treated with DSP .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP.


( E ) HEK293T cells were harvested and treated with DSP .

(E) HEK293T cells were harvested and treated with DSP.


( F ) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h . After treatment with DSP , cell lysates were subjected to IP analysis .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis.


( G ) Atg14F / F ( undeleted ) or Atg14Δ / Δ ( deleted ) MEFs were harvested and treated with DSP . * ( E ) and * * ( G ) indicate the positions of the immunoglobulin light and heavy chains , respectively . aa , amino acid ; DSP , dithiobis ( succinimidyl propionate ) ; IP , immunoprecipitation ; MEFs , mouse embryonic fibroblasts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.


( A ) Immunoblot analysis of Atg proteins in the indicated MEFs .

(A) Immunoblot analysis of Atg proteins in the indicated MEFs.


( B ) m5 ‐ 7 cells were cultured in regular medium containing 10 ng / ml Dox for 8 days , and then cultured in the absence of Dox for a further 2 days .

O O O O O O O O O O O O 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) m5‐7 cells were cultured in regular medium containing 10 ng/ml Dox for 8 days, and then cultured in the absence of Dox for a further 2 days.


( C , D ) m5 ‐ 7 cells were cultured in regular medium with or without 10 ng / ml Dox for 4 days , and then cultured in the presence of 50 μg / ml CHX for the indicated time periods ( C ) . mRNA levels of the indicated genes were measured by quantitative PCR following the 4 ‐ day Dox treatment . Data represent mean ± s . e . ( * P0 . 05 ) ( D ) . CHX , cycloheximide ; Dox , doxycycline ; MEFs , mouse embryonic fibroblasts ; mRNA , messenger RNA ; WT , wild ‐ 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR 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) m5‐7 cells were cultured in regular medium with or without 10 ng/ml Dox for 4 days, and then cultured in the presence of 50 μg/ml CHX for the indicated time periods (C). mRNA levels of the indicated genes were measured by quantitative PCR following the 4‐day Dox treatment. Data represent mean±s.e. (*P0.05) (D). CHX, cycloheximide; Dox, doxycycline; MEFs, mouse embryonic fibroblasts; mRNA, messenger RNA; WT, wild‐type.


( B , C ) HEK293T cells were co ‐ transfected with the indicated constructs . Cell lysates were analysed by IP . IP , immunoprecipitation ; ND , not determined .

O O O O O O O O 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, C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were analysed by IP. IP, immunoprecipitation; ND, not determined.


( A ) WT MEFs , Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h . * indicates nonspecific immunoreactive bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

(A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands.


( B , C ) Atg16L1 KO MEFs stably expressing GFP - ULK1 and either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM ( C ) or starvation medium ( B , C ) for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP , anti ‐ Atg16L1 and anti ‐ LC3 antibodies . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) . The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods . Data represent mean ± s . e . ( * P0 . 001 , analysis of variance followed by Bonferroni / Dunn post hoc test ) . Scale bar , 10 μm , and 2 μm in inset .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) Atg16L1 KO MEFs stably expressing GFP-ULK1 and either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM (C) or starvation medium (B,C) for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP, anti‐Atg16L1 and anti‐LC3 antibodies. Arrowheads indicate the perinuclear localization of Atg16L1(1-230). The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods. Data represent mean±s.e. (*P0.001, analysis of variance followed by Bonferroni/Dunn post hoc test). Scale bar, 10 μm, and 2 μm in inset.


( D , E ) Cells stably expressing GFP - LC3 were cultured in regular DMEM , starvation medium or starvation medium in the presence of 0 . 2 μM WM or 100 nM BafA1 for 6 h . Total cellular GFP - LC3 signals were analysed by flow cytometry . Representative FACS data were shown ( D ) . The geometric mean of fluorescence intensity was determined . Values are expressed as a percentage of the mean of control cells cultured in regular DMEM . Data represent mean ± s . e . ( * P0 . 05 ) ( E ) . BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; KO , knockout ; MEFs , mouse embryonic fibroblasts ; NS , not significant ; WM , wortmannin ; WT , wild ‐ 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

(D, E) Cells stably expressing GFP-LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP-LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.


( A ) Atg16L1 KO MEFs stably expressing either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and subjected to immunofluorescence microscopy using anti ‐ Atg16L1 antibody . Note that full ‐ length Atg16L1 and Atg16L1 ( 1 - 230 ) , but not Atg16L1Δ ( 230 - 300 ) , showed punctate structures under starvation conditions ( inset ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and subjected to immunofluorescence microscopy using anti‐Atg16L1 antibody. Note that full‐length Atg16L1 and Atg16L1(1-230), but not Atg16L1Δ(230-300), showed punctate structures under starvation conditions (inset).


( B ) FIP200 KO MEFs stably expressing either CFP - Atg16L1 ( 1 - 588 ) , CFP - Atg16L1 ( 1 - 230 ) or CFP - Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP antibody . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FIP200 KO MEFs stably expressing either CFP-Atg16L1(1-588), CFP-Atg16L1(1-230) or CFP-Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP antibody. Arrowheads indicate the perinuclear localization of Atg16L1(1-230).


( C , D ) FIP200 KO MEFs stably expressing CFP - Atg16L1 ( 1 - 230 ) were cultured in regular DMEM . Scale bar , 10 μm , and 2 μm in inset .

(C, D) FIP200 KO MEFs stably expressing CFP-Atg16L1(1-230) were cultured in regular DMEM. Scale bar, 10 μm, and 2 μm in inset.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom , Log Rank , p < 0 . 001 , n ≥ 10 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival plot of cohorts of induced Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom, Log Rank, p < 0.001, n ≥ 10).


( b ) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint . Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine ( red arrows ) .

O O O O O O O O O O O O O O O O O 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) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint. Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine (red arrows).


( c ) H & amp ; amp ; E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion . Black arrows indicate individual adenomas . Scale bars = 200 µm .

O O O O O O O O O O O O O O O O O O O O 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) H&amp;E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion. Black arrows indicate individual adenomas. Scale bars = 200 µm.


( d ) Quantification of total tumour numbers per gut in sacrificed Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant increase in the number of tumours per gut ( Mann Whitney , n ≥ 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Quantification of total tumour numbers per gut in sacrificed Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant increase in the number of tumours per gut (Mann Whitney, n ≥ 5).


( a ) H & amp ; amp ; E staining of control and Huwe1 deleted intestinal epithelium . Shortened villi in Huwe1 deficient tissue are indicated . Scale bars = 100 µm . ( b ) BrdU IHC of control and Huwe1 deleted intestinal epithelium . Scale bars = 100 µm . ( c ) PAS staining identifying Goblet cells . No gross changes were observed . Scale bars = 100 µm . ( d ) Lysozyme staining ( Paneth cell marker ) of control and Huwe1 deleted small intestine . Note the occurrence of lysozyme positive cells away from the crypt base ( black arrows ) . Scale bars = 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) H&amp;E staining of control and Huwe1 deleted intestinal epithelium. Shortened villi in Huwe1 deficient tissue are indicated. Scale bars = 100 µm. (b) BrdU IHC of control and Huwe1 deleted intestinal epithelium. Scale bars = 100 µm. (c) PAS staining identifying Goblet cells. No gross changes were observed. Scale bars = 100 µm. (d) Lysozyme staining (Paneth cell marker) of control and Huwe1 deleted small intestine. Note the occurrence of lysozyme positive cells away from the crypt base (black arrows). Scale bars = 100 µm.


( e ) Dual Periodic acid - Schiff / Alcian blue staining to identify Paneth cell secretory vesicles ( light blue / pink - marked with black arrows ) and goblet cells ( dark blue / purple - marked with red arrows ) . Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines ( inset - black arrows ) . Scale bars = 100µm .

(e) Dual Periodic acid-Schiff / Alcian blue staining to identify Paneth cell secretory vesicles (light blue / pink - marked with black arrows) and goblet cells (dark blue / purple - marked with red arrows). Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines (inset - black arrows). Scale bars = 100µm.


( f ) MMP7 staining of control and Huwe1 deleted small intestine . Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines . Scale bars = 100µm .

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(f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.


( a ) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines . Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) QRT - PCR analysis of Myc expression in control and Huwe1 deficient intestines .

(b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines.


( c ) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice . Scale bars = 50 µm .

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(c) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice. Scale bars = 50 µm.


( d ) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MYC protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

(d) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MYC protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( e ) QRT - PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice .

O O O O O O O O O O O O O O O O O O O O O

(e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc , Log Rank , p < 0 . 001 , n ≥ 16 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kaplan-Meier survival plot of cohorts of induced Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc, Log Rank, p < 0.001, n ≥ 16).


( b ) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc . Mann Whitney , n ≥ 10 ) .

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(b) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc. Mann Whitney, n ≥ 10).


( c ) Kaplan - Meier survival plot of cohorts of induced AhCre - ERT Apc Pten and AhCre - ERT Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC led to a significant reduction in survival ( Log Rank , p < 0 . 001 , n = 14 vs 21 ) .

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(c) Kaplan-Meier survival plot of cohorts of induced AhCre-ERT Apc Pten and AhCre-ERT Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC led to a significant reduction in survival (Log Rank, p < 0.001, n = 14 vs 21).


( d ) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice ( Mann Whitney , n = 9 vs 19 ) .

(d) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice (Mann Whitney, n = 9 vs 19).


( a ) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells . Band intensity relative to βactin displayed under each lane ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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(a) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells. Band intensity relative to βactin displayed under each lane (Mann-Whitney, p = 0.04, n = 3 vs 3).


( b ) γ - H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells . Scale bars = 50 µm .

(b) γ-H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells. Scale bars = 50 µm.


( c ) γ - H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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(c) γ-H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells (Mann-Whitney, p = 0.04, n = 3 vs 3).


( d ) γ - H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours , note increased positivity in Huwe1 deficient tumours . Scale bars = 50 µm .

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(d) γ-H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours, note increased positivity in Huwe1 deficient tumours. Scale bars = 50 µm.


( a ) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7 . 5mg / kg cisplatin . Arrows identify caspase 3 positive cells . Scale bars = 50 µm . ( b ) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells ( Mann - Whitney , n = 3 vs 5 ) .

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(a) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7.5mg/kg cisplatin. Arrows identify caspase 3 positive cells. Scale bars = 50 µm. (b) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells (Mann-Whitney, n = 3 vs 5).


( c ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not . Note the significant increase in mutational burden in HUWE1 mutated tumours ( Mann - Whitney , p = 0 . 0002 , n ≥ 15 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not. Note the significant increase in mutational burden in HUWE1 mutated tumours (Mann-Whitney, p = 0.0002, n ≥ 15).


( d ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status . Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate ( and HUWE1 mutation itself ) is not due to silencing of MLH1 ( Mann - Whitney , p = 0 . 0007 , n ≥ 11 ) .

(d) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not grouped according to MLH1 status. Note that the increased mutation rate is found primarily in tumours where MLH1 is not silenced indicating increased mutation rate (and HUWE1 mutation itself) is not due to silencing of MLH1 (Mann-Whitney, p = 0.0007, n ≥ 11).


( e ) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels . Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy . ( f ) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels . Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy .

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(e) Survival analysis of colorectal cancer patients treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients respond significantly better to chemotherapy. (f) Survival analysis of colorectal cancer patients not treated with adjuvant chemotherapy divided by HUWE1 expression levels. Note the lowest HUWE1 expressing quartile of patients do not survive significantly longer than those expressing higher levels of HUWE1 if not treated with chemotherapy.


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Log Rank , p < 0 . 001 , n ≥ 15 ) .

(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc and Lgr5 Apc Huwe1 mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Log Rank, p < 0.001, n ≥ 15).


( c ) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) . ( d ) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours . Scale bars = 200 µm ( low magnification ) , 100 µm ( high magnification inset ) .

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(c) Lysozyme IHC demonstrating increased lysozyme positive cell numbers in tumours from Lgr5 Apc Huwe1 mice. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset). (d) OLFM4 IHC demonstrating expanded stem cell population in Lgr5 Apc Huwe1 tumours. Scale bars = 200 µm (low magnification), 100 µm (high magnification inset).


( a ) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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(a) MCL1 Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MCL1 protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) Kaplan - Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice . Deletion of one copy of Mcl1 led to a significant increase in survival of these animals ( Log Rank , p = 0 . 0052 , n = 9 vs 10 ) .

(b) Kaplan-Meier survival plot of cohorts of induced Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1 mice. Deletion of one copy of Mcl1 led to a significant increase in survival of these animals (Log Rank, p = 0.0052, n = 9 vs 10).


( c ) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice . Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion ( Mann - Whitney , p = 0 . 0281 , n = 8 v 8 ) .

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(c) Ratio of adenoma / indolent lesions observed in Lgr5 Apc Huwe1 and Lgr5 Apc Huwe1 Mcl1het mice. Note the decreased ration of adenoma / indolent lesions observed upon Mcl1 deletion (Mann-Whitney, p=0.0281, n = 8 v 8).


( A ) Ookinetes sandwiched between two hydrogel surfaces .

(A) Ookinetes sandwiched between two hydrogel surfaces.


( B ) Percentage of ookinetes moving for more than one parasite length within the time of observation ( 5 - 10 min ) if sandwiched between soft , medium ( med ) or stiff hydrogel or glass . Bars represent the mean and error bars the standard deviation of at least three independent experiments . ( C ) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B .

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(B) Percentage of ookinetes moving for more than one parasite length within the time of observation (5-10 min) if sandwiched between soft, medium (med) or stiff hydrogel or glass. Bars represent the mean and error bars the standard deviation of at least three independent experiments. (C) Speed of motile ookinetes on substrates of different stiffnesses as indicated in panel B.


( D ) Percentage of motile ookinetes after 20 - 26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in ( B ) . Shown is the average ± S . D . from at least two independent experiments . ( E ) Speed of motile ookinetes after 20 - 26 hours of incubation between hydrogels . Speed on glass was not analyzed ( n . a . ) due to the low fraction of motile ookinetes at this timepoint .

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(D) Percentage of motile ookinetes after 20-26 hours of incubation between hydrogels compared to parasites directly imaged after setting up the experiment as shown in (B). Shown is the average ± S.D. from at least two independent experiments. (E) Speed of motile ookinetes after 20-26 hours of incubation between hydrogels. Speed on glass was not analyzed (n.a.) due to the low fraction of motile ookinetes at this timepoint.


( F ) Migration patterns of ookinetes . Image : Overlay of tracked migration paths ( black ) and DIC image showing ookinetes at the end of the recorded image sequence . Graph : Quantitative analysis of the indicated different migration patterns on soft , medium ( med ) and stiff hydrogels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Migration patterns of ookinetes. Image: Overlay of tracked migration paths (black) and DIC image showing ookinetes at the end of the recorded image sequence. Graph: Quantitative analysis of the indicated different migration patterns on soft, medium (med) and stiff hydrogels.


( G ) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints .

(G) Mean square displacement of ookinetes moving on hydrogels of different stiffnesses at two timepoints.


( A ) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel .

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(A) Infected salivary glands were sandwiched between a soft PA hydrogel and a glass coverslip inducing sporozoites to move into the hydrogel.


( B ) Overlay of sporozoite tracks ( black ) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence . Scale bar , 20 µm .

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(B) Overlay of sporozoite tracks (black) and DIC image showing sporozoites inside a soft hydrogel at the end of a recorded image sequence. Scale bar, 20 µm.


( C ) Speed of WT sporozoites moving in the skin in vivo ( Douglas et al , 2018b ) and in soft hydrogels with small or large pores .

(C) Speed of WT sporozoites moving in the skin in vivo (Douglas et al, 2018b) and in soft hydrogels with small or large pores.


( D , E ) Speed of indicated mutant and control ( Ctrl ) parasites in hydrogels with small ( D ) and large ( E ) pores .

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(D, E) Speed of indicated mutant and control (Ctrl) parasites in hydrogels with small (D) and large (E) pores.


( F , G ) Speed of drug - treated and control parasites in hydrogels with small ( F ) and large ( G ) pores . Data information : Note the faster speed in gels with larger pores . Data from at least two independent experiments . Numbers above bars indicate the number of sporozoites analyzed . Significance determined by Mann - Whitney test . Box - and - whisker plots ( C - G ) depict the 25 % quantile , median , 75 % quantile and nearest observations within 1 . 5 times the interquartile range ( whiskers ) . Outliers beyond this range are shown as black dots .

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(F, G) Speed of drug-treated and control parasites in hydrogels with small (F) and large (G) pores. Data information: Note the faster speed in gels with larger pores. Data from at least two independent experiments. Numbers above bars indicate the number of sporozoites analyzed. Significance determined by Mann-Whitney test. Box-and-whisker plots (C-G) depict the 25% quantile, median, 75% quantile and nearest observations within 1.5 times the interquartile range (whiskers). Outliers beyond this range are shown as black dots.


( A ) Percentage of motile sporozoites on endothelial cells ( LSECs ) versus glass . Shown is average ± S . D . from three independent experiments . Significance determined using an unpaired t - test .

O O O O O O O O O O O O O O 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) Percentage of motile sporozoites on endothelial cells (LSECs) versus glass. Shown is average ± S.D. from three independent experiments. Significance determined using an unpaired t-test.


( B ) Left : Merged fluorescence images showing sporozoites on cultured HFF cells . Actin filaments of HFF cells were stained with phalloidin ( shown in red ) , nuclei were stained with Hoechst ( blue ) and sporozoites expressed GFP ( green ) . Scale bar , 50 µm . Right : Maximum projection of sporozoites moving for 3 min on cultured HFF cells . Scale bar , 50 µm .

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(B) Left: Merged fluorescence images showing sporozoites on cultured HFF cells. Actin filaments of HFF cells were stained with phalloidin (shown in red), nuclei were stained with Hoechst (blue) and sporozoites expressed GFP (green). Scale bar, 50 µm. Right: Maximum projection of sporozoites moving for 3 min on cultured HFF cells. Scale bar, 50 µm.


( C ) Percentage of motile sporozoites on confluent layers of endothelial cells ( HUVEC ) , fibroblasts ( HFF ) and on glass . Shown is average ± S . D . from two independent experiments . No significant differences as determined by One - way analysis of variance with Bonferroni ' s Multiple Comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage of motile sporozoites on confluent layers of endothelial cells (HUVEC), fibroblasts (HFF) and on glass. Shown is average ± S.D. from two independent experiments. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


( D ) Sporozoite on planar uncoated hydrogels .

(D) Sporozoite on planar uncoated hydrogels.


( E ) Overlay of sporozoite tracks ( black ) and DIC image showing sporozoites on a hydrogel . Scale bar , 50 µm . ( F ) Percentage of motile sporozoites on soft , medium ( med ) or stiff hydrogel or glass 30 min after activation with BSA . Shown is the average ± S . D . from at least three independent experiments . Significance determined by One - way analysis of variance with Bonferroni ' s Multiple Comparison test .

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(E) Overlay of sporozoite tracks (black) and DIC image showing sporozoites on a hydrogel. Scale bar, 50 µm.(F) Percentage of motile sporozoites on soft, medium (med) or stiff hydrogel or glass 30 min after activation with BSA. Shown is the average ± S.D. from at least three independent experiments. Significance determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


( G ) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA . All sporozoites moving for more than one parasite length within the time of observation were analyzed , even if they stopped moving or detached during imaging . Significance determined by Kruskal - Wallis test with Dunn ' s Multiple Comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Migration persistance as determined by the number of circles performed within 100 s on hydrogels of different stiffnesses and glass 30 min after activation with BSA. All sporozoites moving for more than one parasite length within the time of observation were analyzed, even if they stopped moving or detached during imaging. Significance determined by Kruskal-Wallis test with Dunn's Multiple Comparison test.


( H ) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses . Note that not all sporozoites analyzed in ( G ) complied with that requirement . To be able to analyze a high number of sporozoites , movies were taken 10 to 45 min after activation with BSA . Speed of sporozoites on soft hydrogels was not analyzed ( n . a . ) due to the low fraction of motile sporozoites on these hydrogels . No significant differences as determined by One - way analysis of variance with Bonferroni ' s Multiple Comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Speed of sporozoites moving consistently for at least 60 s on substrates of different stiffnesses. Note that not all sporozoites analyzed in (G) complied with that requirement. To be able to analyze a high number of sporozoites, movies were taken 10 to 45 min after activation with BSA. Speed of sporozoites on soft hydrogels was not analyzed (n.a.) due to the low fraction of motile sporozoites on these hydrogels. No significant differences as determined by One-way analysis of variance with Bonferroni's Multiple Comparison test.


A The family pedigree , where circles denote female members , squares male members , solid symbols affected members , and white symbols asymptomatic members with normal physical exam ; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage . The pictures show scapular winging which is a consistent clinical sign in affected individuals .

A The family pedigree, where circles denote female members, squares male members, solid symbols affected members, and white symbols asymptomatic members with normal physical exam; the dots indicate heterozygous carriers and double line denotes a consanguineous marriage. The pictures show scapular winging which is a consistent clinical sign in affected individuals.


B Hematoxylin and eosin staining ( H & amp ; amp ; E ) of skeletal muscle from patient II . 1 shows histological features of moderate to severe dystrophic pattern . ( Scale bar , 50 μm ) .

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B Hematoxylin and eosin staining (H&amp;E) of skeletal muscle from patient II.1 shows histological features of moderate to severe dystrophic pattern. (Scale bar, 50 μm).


C T1 - weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles , equally affecting posterior and anterior compartment , with relative sparing of the rectus femoris , sartorius and gracilismuscles until late stages ( 4 , 10 and 11 , respectively ) . Strikingly , the fatty tissue is located in the internal parts of almost all the affected muscles in thigh ( 1 , 2 , 3 , 5 - 9 ) , while the external regions are spared . At calf level , only the gastrocnemius medialis muscle ( 12 ) shows this pattern , while the soleus ( 13 ) is diffusely involved . Patient II . 2 ( PII . 2 ) shows late - stage thigh muscles with an unusual involvement of the tibialis posteriormuscle ( 14 ) in the lower leg .

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C T1-weighted MRI axial images at thigh and calf levels show that the fatty degeneration is more prominent in thigh muscles, equally affecting posterior and anterior compartment, with relative sparing of the rectus femoris, sartorius and gracilismuscles until late stages (4, 10 and 11, respectively). Strikingly, the fatty tissue is located in the internal parts of almost all the affected muscles in thigh (1, 2, 3, 5-9), while the external regions are spared. At calf level, only the gastrocnemius medialis muscle (12) shows this pattern, while the soleus (13) is diffusely involved. Patient II.2 (PII.2) shows late-stage thigh muscles with an unusual involvement of the tibialis posteriormuscle (14) in the lower leg.


A Muscle sections show variable labeling using an antibody against glycosylated α - dystroglycan ( αDG - IIH6 ) , whereas labeling using antibodies against α - dystroglycan core protein ( αDG - Core ) , β - dystroglycan ( βDG ) and laminin - 2 is similar to control ( Scale bar , 100 μm ) .

A Muscle sections show variable labeling using an antibody against glycosylated α-dystroglycan (αDG-IIH6), whereas labeling using antibodies against α-dystroglycan core protein (αDG-Core), β-dystroglycan (βDG) and laminin-2 is similar to control (Scale bar, 100 μm).


B Western blots and ligand - overlay ( O / L ) of wheat - germ agglutinin - enriched muscle and fibroblasts lysates from PII . 2 , PII . 5 , the healthy sibling ( HII . 3 ) and healthy controls ( Ctr , Ctr1 , and Ctr2 ) ( Muscle : 250μg protein / lane , Fibroblasts : 800μg / lane ) . In muscle , expression of αDG - IIH6 is reduced but the αDG - Core shows similar expression as controls , with a slight reduction in molecular weight ; laminin overlay assay detected some binding activity but was diminished compared to controls , whereas agrin - binding activity showed no difference with controls . In fibroblasts , both αDG - IIH6 and αDG - Core expression and laminin - binding activity were normal , suggesting that unlike other muscular dystrophies , α - dystroglycan glycosylation defect is muscle - specific in POGLUT1 patients .

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B Western blots and ligand-overlay (O/L) of wheat-germ agglutinin-enriched muscle and fibroblasts lysates from PII.2, PII.5, the healthy sibling (HII.3) and healthy controls (Ctr, Ctr1, and Ctr2) (Muscle: 250μg protein/lane, Fibroblasts: 800μg/lane). In muscle, expression of αDG-IIH6 is reduced but the αDG-Core shows similar expression as controls, with a slight reduction in molecular weight; laminin overlay assay detected some binding activity but was diminished compared to controls, whereas agrin-binding activity showed no difference with controls. In fibroblasts, both αDG-IIH6 and αDG-Core expression and laminin-binding activity were normal, suggesting that unlike other muscular dystrophies, α-dystroglycan glycosylation defect is muscle-specific in POGLUT1 patients.


C No ultrastructural alterations are observed in muscle by electron microscopy ( 6 - 7 fields were captured from PII . 1 , PII . 3 , and PII . 5 ) . Note normal basement membrane compactation ( arrowheads ) . Original magnification : 26 , 700 .

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C No ultrastructural alterations are observed in muscle by electron microscopy (6-7 fields were captured from PII.1, PII.3, and PII.5). Note normal basement membrane compactation (arrowheads). Original magnification: 26,700.


A Protein O - glucosyltransferase activity of wild - type ( WT ) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat ( hFIX - EGF ) . Wild - type shows higher O - glucosyltransferase activity than D233E , and this activity is dependent on the concentration of the acceptor substrate hFIX - EGF repeat ( left ) and on the concentration of donor substrate UDP - glucose ( UDP - Glc ) ( right ) . Values indicate mean ± SEM from three independent assays .

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A Protein O-glucosyltransferase activity of wild-type (WT) or D233E mutant POGLUT1 protein toward human factor IX EGF repeat (hFIX-EGF). Wild-type shows higher O-glucosyltransferase activity than D233E, and this activity is dependent on the concentration of the acceptor substrate hFIX-EGF repeat (left) and on the concentration of donor substrate UDP-glucose (UDP-Glc) (right). Values indicate mean ± SEM from three independent assays.


B O - glucosyltransferase activity towards five different single EGF repeats from mouse Notch1 . Wild - type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E . The Y - axis shows the normalized activity relative to wild - type POGLUT1 . EGF repeats were at 10 μM . Values indicate mean ± SEM from three independent assays .

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B O-glucosyltransferase activity towards five different single EGF repeats from mouse Notch1. Wild-type POGLUT1 shows higher activity toward all EGF repeats tested than POGLUT1D233E. The Y-axis shows the normalized activity relative to wild-type POGLUT1. EGF repeats were at 10 μM. Values indicate mean ± SEM from three independent assays.


C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC . hFIX - EGF repeat was incubated with wild - type or D233E mutant POGLUT1 and donor substrate , UDP - Glc or UDP - xylose ( UDP - Xyl ) , at 37ºC overnight . Values on top of the peaks indicate the measured masses . Addition of Glc ( 162 Da ) or xylose ( 132 Da ) to hFIX - EGF ( 5696 . 2 Da ) by wild - type POGLUT1 caused a shift to an earlier retention time ( left ) . Similarly , the products exhibited a similar shift after incubation with POGLUT1D233E ( right ) . These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX - EGF repeats and thus has residual enzymatic activity .

O O O O O O O O O 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-CONTROLLED_VAR O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

C Elution profiles of the POGLUT1 reaction products on reverse phase HPLC. hFIX-EGF repeat was incubated with wild-type or D233E mutant POGLUT1 and donor substrate, UDP-Glc or UDP-xylose (UDP-Xyl), at 37ºC overnight. Values on top of the peaks indicate the measured masses. Addition of Glc (162 Da) or xylose (132 Da) to hFIX-EGF (5696.2 Da) by wild-type POGLUT1 caused a shift to an earlier retention time (left). Similarly, the products exhibited a similar shift after incubation with POGLUT1D233E (right). These results indicate that POGLUT1D233E can add a single glucose or xylose to hFIX-EGF repeats and thus has residual enzymatic activity.


A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain ( NICD ) in patients .

A Western blot of muscle homogenates shows reduced expression of Notch1 intracellular domain (NICD) in patients.


B - C qRT - PCR on RNA extracted from skeletal - muscle shows that expression of HES1 ( B ) and PAX7 ( C ) was significantly lower in D233E patients compared to healthy controls . Mean ± SEM , Student ´ s t - test ( B ) and Mann - Whitney U test ( C ) .

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B-C qRT-PCR on RNA extracted from skeletal-muscle shows that expression of HES1 (B) and PAX7 (C) was significantly lower in D233E patients compared to healthy controls. Mean ± SEM, Student´s t-test (B) and Mann-Whitney U test (C).


D PAX7 + cells in skeletal muscle sections , demonstrating that satellite cells are less abundant in D233E patients than in controls ( n = 3 muscles with 10 - 15 fields analyzed per muscle ) . Mean ± SEM ; Mann - Whitney U test ; Scale bar , 50 μm .

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D PAX7+ cells in skeletal muscle sections, demonstrating that satellite cells are less abundant in D233E patients than in controls (n = 3 muscles with 10-15 fields analyzed per muscle). Mean ± SEM; Mann-Whitney U test; Scale bar, 50 μm.


A - A " Staining of Drosophila indirect flight muscles at 25 % pupal development with 22C10 antibody ( myotube marker ) and anti - Twist antibody ( myoblast marker ) indicates that in wild - type animals , indirect flight muscles are formed from 6 myotubes , and a large number of Twist + myoblasts are present . B - B " rumi79 / 79 mutants raised at 18C show no obvious defects in myoblast number or myotube morphology . C - D " rumi79 / 79 mutants exhibit decreased number of myoblasts at 25 ° - 30 ° C and either an incomplete set of short myotubes when raised at 25C or aberrant morphology of myotubes when raised at 30C . E - E " rumi79 / 79 animals overexpressing FLAG - tagged POGLUT1WT in the muscle compartment using Mef2 - GAL4 and raised at 25C during the pupal stage show a rescue of the rumi79 / 79 muscle phenotype . F - G " Overexpression of FLAG - tagged POGLUT1D233E only partially rescues the rumi79 / 79 muscle phenotype , with some variation in the myotube length and the number of restored Twist + cells ( compare G and F ) . Note that the number of Twist + cells restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT . Scale bar in A ' is 50 µm and applies to A - G " . H Quantification of myotube lengths in rumi79 / 79 mutants with or without POGLUT1 expression indicates a weak rescue of the phenotypes by POGLUT1D233E compared to POGLUT1WT . Mean ± standard deviation is shown . The differences in myotube lengths are statistically significant ( n = 22 - 54 ) . Mean ± standard deviation is shown ; One - way Anova with Bonferroni ´ s multiple comparisons test .

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A-A" Staining of Drosophila indirect flight muscles at 25% pupal development with 22C10 antibody (myotube marker) and anti-Twist antibody (myoblast marker) indicates that in wild-type animals, indirect flight muscles are formed from 6 myotubes, and a large number of Twist+myoblasts are present.B-B" rumi79/79 mutants raised at 18C show no obvious defects in myoblast number or myotube morphology.C-D" rumi79/79 mutants exhibit decreased number of myoblasts at 25°-30°C and either an incomplete set of short myotubes when raised at 25C or aberrant morphology of myotubes when raised at 30C.E-E" rumi79/79 animals overexpressing FLAG-tagged POGLUT1WT in the muscle compartment using Mef2-GAL4 and raised at 25C during the pupal stage show a rescue of the rumi79/79 muscle phenotype.F-G" Overexpression of FLAG-tagged POGLUT1D233E only partially rescues the rumi79/79 muscle phenotype, with some variation in the myotube length and the number of restored Twist+cells (compare G and F). Note that the number of Twist+cells restored by POGLUT1D233E is much smaller than that restored by POGLUT1WT. Scale bar in A' is 50 µm and applies to A-G".H Quantification of myotube lengths in rumi79/79 mutants with or without POGLUT1 expression indicates a weak rescue of the phenotypes by POGLUT1D233E compared to POGLUT1WT. Mean ± standard deviation is shown. The differences in myotube lengths are statistically significant (n = 22-54). Mean ± standard deviation is shown; One-way Anova with Bonferroni´s multiple comparisons test.


A - F Proliferation assays examined proliferating ( PAX7 + MyoD + , arrows ) , self - renewing ( PAX7 + MyoD − , open arrows ) , and differentiating ( PAX7 − MyoD + , arrowheads ) cells from D233E patients ( n = 2 ) , healthy controls ( n = 3 ) and disease controls ( n = 3 ) at 1 , 4 , and 8 days growing in proliferation medium . ( A ) shows representative images from day 8 . The number of patientmyoblasts is lower than that of controls at all three time points , indicating a slower proliferation rate in patientmyoblasts ( B ) , and accordingly , the percentage of proliferating cells ( C ) is smaller in the patient . ( D ) The percentage of self - renewing cells is small in patient ´ s culture , reflecting a poor capacity for maintaining the pool of quiescent SCs . In addition , the percentage of PAX7 + cells ( E ) is smaller in patient ´ s culture , similarly to what was found in adult D233E muscle ( shown in Fig 5D ) . ( F ) The percentage of differentiating cells is higher in the patient . We examine proliferation capturing 7 - 12 randomly fields per day and condition ( mean ± SEM is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons test ; Scale bar , 50 µm ) .

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A-F Proliferation assays examined proliferating (PAX7+MyoD+, arrows), self-renewing (PAX7+ MyoD−, open arrows), and differentiating (PAX7− MyoD+, arrowheads) cells from D233E patients (n = 2), healthy controls (n = 3) and disease controls (n = 3) at 1, 4, and 8 days growing in proliferation medium. (A) shows representative images from day 8. The number of patientmyoblasts is lower than that of controls at all three time points, indicating a slower proliferation rate in patientmyoblasts (B), and accordingly, the percentage of proliferating cells (C) is smaller in the patient. (D) The percentage of self-renewing cells is small in patient´s culture, reflecting a poor capacity for maintaining the pool of quiescent SCs. In addition, the percentage of PAX7+ cells (E) is smaller in patient´s culture, similarly to what was found in adult D233E muscle (shown in Fig 5D). (F) The percentage of differentiating cells is higher in the patient. We examine proliferation capturing 7-12 randomly fields per day and condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).


G - I When myoblasts started to be confluent , the proliferation medium was replaced with differentiation medium , and the myoblasts started to fuse yielding myotubes , which were analyzed after four days of differentiation . ( H ) The fusion index , which measures the percentage of nuclei in myotubes with respect to the total number of nuclei in myogenic cells , was higher in D233E cultures than in controls . ( I ) Besides , myogenin showed increased expression in D233E myoblast cultures compared to controls . The data support that differentiation process is facilitated in patient ´ s muscle . Data was collected from 3 - 10 randomly chosen areas per condition ( mean ± SEM is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons test ( H ) and One - way Anova with Bonferroni ´ s multiple comparisons test ( I ) ; Scale bar , 50 µm ) .

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G-I When myoblasts started to be confluent, the proliferation medium was replaced with differentiation medium, and the myoblasts started to fuse yielding myotubes, which were analyzed after four days of differentiation. (H) The fusion index, which measures the percentage of nuclei in myotubes with respect to the total number of nuclei in myogenic cells, was higher in D233E cultures than in controls. (I) Besides, myogenin showed increased expression in D233E myoblast cultures compared to controls. The data support that differentiation process is facilitated in patient´s muscle. Data was collected from 3-10 randomly chosen areas per condition (mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test (H) and One-way Anova with Bonferroni´s multiple comparisons test (I); Scale bar, 50 µm).


A - C We overexpressed GFP - tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts . ( B ) We analyzed the proliferation at 1 , 3 and 5 days , and proliferation rate in patient - LV - NICD - GFP reached the level observed in control - LV - GFP , which were higher than in patient - LV - GFP . ( C ) However , the percentage of PAX7 + cells in patient - LV - NICD - GFP showed the same reduced value as in patient - LV - GFP , which were significantly lower than in control - LV - GFP . The majority of control myoblasts were proliferating cells ( PAX7 + MyoD + , arrows ) , with no quiescent cells and few differentiating cells detected . Data were collected from n = 2 independent assays ( 5 - 8 images per condition ) . Mean ± SEM is shown ; One - way Anova with Tukey multiple comparisions test ( B ) and Kruskal - Wallis with Dunn ´ s multiple comparisons test ( C ) ; Scale bar , 50 µm ) .

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A-C We overexpressed GFP-tagged NICD in immortalized primary myoblasts to rescue the features displayed by immortalized D233E myoblasts. (B) We analyzed the proliferation at 1, 3 and 5 days, and proliferation rate in patient-LV-NICD-GFP reached the level observed in control-LV-GFP, which were higher than in patient-LV-GFP. (C) However, the percentage of PAX7+cells in patient-LV-NICD-GFP showed the same reduced value as in patient-LV-GFP, which were significantly lower than in control-LV-GFP. The majority of control myoblasts were proliferating cells (PAX7+ MyoD+, arrows), with no quiescent cells and few differentiating cells detected. Data were collected from n = 2 independent assays (5-8 images per condition). Mean ± SEM is shown; One-way Anova with Tukey multiple comparisions test (B) and Kruskal-Wallis with Dunn´s multiple comparisons test (C); Scale bar, 50 µm).


D - F As previously described in patient ' s primary myoblasts , patient - LV - GFPmyoblasts showed a striking facilitated differentiation compared to control - LV - GFP - myoblasts . This phenotype fully disappeared in patient - LV - GFP - NICD myoblasts , as quantification of the fusion index ( E ) , and myogenin expression ( F ) demonstrated . The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients . Data were collected from n = 3 independent assays ( 13 - 17 images per condition ) . Mean ± SEM is shown ; Kruskal - Wallis with Dunn ´ s multiple comparisons test ; Scale bar , 50 µm ) .

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D-F As previously described in patient's primary myoblasts, patient-LV-GFPmyoblasts showed a striking facilitated differentiation compared to control-LV-GFP-myoblasts. This phenotype fully disappeared in patient-LV-GFP-NICD myoblasts, as quantification of the fusion index (E), and myogenin expression (F) demonstrated. The data support a pathogenic role for Notch inhibition on the altered differentiation in our patients. Data were collected from n = 3 independent assays (13-17 images per condition). Mean ± SEM is shown; Kruskal-Wallis with Dunn´s multiple comparisons test; Scale bar, 50 µm).


A Immortalized primary myoblasts from a healthy control showed a progressive pattern of α - dystroglycan glycosylation during five days of differentiation ( D0 - D5 ) , as has been previously described by Goddeeris et al . Immortalized primary myoblasts from patients displayed an irregular pattern of glycosylation , with a final level of glycosylated α - dystroglycan that was lower than control .

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A Immortalized primary myoblasts from a healthy control showed a progressive pattern of α-dystroglycan glycosylation during five days of differentiation (D0-D5), as has been previously described by Goddeeris et al. Immortalized primary myoblasts from patients displayed an irregular pattern of glycosylation, with a final level of glycosylated α-dystroglycan that was lower than control.


B α - dystroglycan glycosylation of infected immortalized myoblasts from patient - LV - NICD - GFP at differentiation day 5 ( D5 ) showed a striking reduction , in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression . α - dystroglycan in myoblasts from patient - LV - GFP showed a reduction of glycosylation compared to control - LV - GFP at D5 , similar to the results obtained in the adult muscle from patients ( Fig . 2 ) .

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B α-dystroglycan glycosylation of infected immortalized myoblasts from patient-LV-NICD-GFP at differentiation day 5 (D5) showed a striking reduction, in line with the strong inhibition of myotubes formation and differentiation induced by NICD overexpression. α-dystroglycan in myoblasts from patient-LV-GFP showed a reduction of glycosylation compared to control-LV-GFP at D5, similar to the results obtained in the adult muscle from patients (Fig. 2).


C - D C2C12 myogenic cells showed a reduced level of glycosylated α - dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium . This indicates that reducing Notch signaling can affect α - dystroglycan glycosylation in wild - type C2C12 myoblasts cells , which do not harbor any known mutations in α - dystroglycan glycosyltransferases or Poglut1 .

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C-D C2C12 myogenic cells showed a reduced level of glycosylated α-dystroglycan when the Notch signaling inhibitor DAPT or LY3039478 was added to the differentiation medium. This indicates that reducing Notch signaling can affect α-dystroglycan glycosylation in wild-type C2C12 myoblasts cells, which do not harbor any known mutations in α-dystroglycan glycosyltransferases or Poglut1.


E - F qRT - PCR experiments show that upon DAPT or LY3039478 treatment , a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation .

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E-F qRT-PCR experiments show that upon DAPT or LY3039478 treatment, a remarkable decrease in Hes1 mRNA is induced in C2C12 cells during differentiation.


K - P Realtime qRT - PCR revealed the expression profiles of four zebrafish chs genes in different tissues ( K ) , different gut sections ( L ) , and different developmental stages ( M - P ) . One representative example of three repeat experiments is shown . Data were the mean ± SEM of n = 3 technical replicates . n . s . no significant , no label P < 0 . 05 by student ' s t - test .

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K-P Realtime qRT-PCR revealed the expression profiles of four zebrafish chs genes in different tissues (K), different gut sections (L), and different developmental stages (M-P). One representative example of three repeat experiments is shown. Data were the mean ± SEM of n=3 technical replicates. n.s. no significant, no label P < 0.05 by student's t-test.


F - M Compared with wild - types ( F - I ) , mutants ( J - M ) completely lost the CM ( red ) in the gut of 7dpf larva ( F , J ) , as well as in the intestinal bulb ( G , K ) , middle intestine ( H , L ) and posterior intestine ( I , M ) of adults . Red , SNAP - CBD stain ; blue , DAPI stain ; WT , wild - type ; MU , chs1 - / - mutant .

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F-M Compared with wild-types (F-I), mutants (J-M) completely lost the CM (red) in the gut of 7dpf larva (F, J), as well as in the intestinal bulb (G, K), middle intestine (H, L) and posterior intestine (I, M) of adults. Red, SNAP-CBD stain; blue, DAPI stain; WT, wild-type; MU, chs1-/- mutant.


A , B The survival rates of wild - types and mutants in the first eight days ( A ) and the first six months ( B ) . Two independent batches ( 1 and 2 ) were conducted for each experiment . n , the initial numbers of zebrafish . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by long - rank ( Mantel - Cox ) test . Please see Table EV1 for data on another three batches of independent experiments .

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A, B The survival rates of wild-types and mutants in the first eight days (A) and the first six months (B). Two independent batches (1 and 2) were conducted for each experiment. n, the initial numbers of zebrafish. * P < 0.05, ** P < 0.01, *** P < 0.001 by long-rank (Mantel-Cox) test. Please see Table EV1 for data on another three batches of independent experiments.


E - J 6 / 7 dpf larva mutants were fed with 6 - μm polystyrene beads . The beads jammed in the intestinal bulb and middle intestine ( E , F , H , I ) . In some cases , the beads escaped the bead cord and stuck between villi ( white triangles ) ( E , G , I , J ) . Blue rectangle indicates the intestinal bulb . Right panels were magnification of the red rectangle area in left panels .

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E-J 6/7 dpf larva mutants were fed with 6-μm polystyrene beads. The beads jammed in the intestinal bulb and middle intestine (E, F, H, I). In some cases, the beads escaped the bead cord and stuck between villi (white triangles) (E, G, I, J). Blue rectangle indicates the intestinal bulb. Right panels were magnification of the red rectangle area in left panels.


B Comparison of 3 - month - old wild - types and mutants in the pairwise microbiota compositional dissimilarity ( Jaccard distance ) between the digesta ( niche1 ) and the epithelia ( niche2 ) . This metric represents the radial gradient of the gut bacterial composition . The comparison was done on four taxonomy levels . Data of n = 14 individuals were represented in box ( interquartile range ) and whisker ( min to max ) plots . Individual data points were plotted as black dots . * P < 0 . 05 , * * P < 0 . 01 by the conservative , nonparametric Kruskal - Wallis test . Please see Appendix Fig S10 for another way of data presentation , and Dataset EV1 for the details of the sample information and raw statistics . Note that each 16S - rRNA library contains only one sample from one animal .

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B Comparison of 3-month-old wild-types and mutants in the pairwise microbiota compositional dissimilarity (Jaccard distance) between the digesta (niche1) and the epithelia (niche2). This metric represents the radial gradient of the gut bacterial composition. The comparison was done on four taxonomy levels. Data of n=14 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05, ** P < 0.01 by the conservative, nonparametric Kruskal-Wallis test. Please see Appendix Fig S10 for another way of data presentation, and Dataset EV1 for the details of the sample information and raw statistics. Note that each 16S-rRNA library contains only one sample from one animal.


Comparison of the bacterial community richness of the intestine samples ( S3 - S6 ; including both the digesta and the epithelia ) between six 3 - month - old wild - types and six 3 - month - old mutants . Three alpha diversity metrics were used : Ace ( abundance - based coverage estimator ) , Chao1 Data of n = 6 individuals were represented in box ( interquartile range ) and whisker ( min to max ) plots . Individual data points were plotted as black dots . * P < 0 . 05 by Kruskal - Wallis test .

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Comparison of the bacterial community richness of the intestine samples (S3-S6; including both the digesta and the epithelia) between six 3-month-old wild-types and six 3-month-old mutants. Three alpha diversity metrics were used: Ace (abundance-based coverage estimator), Chao1 Data of n=6 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05 by Kruskal-Wallis test.


E Immunohistochemical detection of BrdU - labeled cells in the intestine bulb ( section S2 ) of two 3 - month - old adult wild - types and two 3 - month - old adult mutants to measure the rate of cell proliferation ( new cells were stained bright green ) and migration . Compared with wild - types ( WT ) , the mutants had more green spots along the villi 2 hours after treatment , and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments . The violin plots ( interquartile range with mean line ) show the relative numbers of green puncta from tip to base of villi , which were calculated from 3 - 5 villi from the raw version of these images . Please see Appendix Fig S19 for the images of more animals and time points .

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E Immunohistochemical detection of BrdU-labeled cells in the intestine bulb (section S2) of two 3-month-old adult wild-types and two 3-month-old adult mutants to measure the rate of cell proliferation (new cells were stained bright green) and migration. Compared with wild-types (WT), the mutants had more green spots along the villi 2 hours after treatment, and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments. The violin plots (interquartile range with mean line) show the relative numbers of green puncta from tip to base of villi, which were calculated from 3-5 villi from the raw version of these images. Please see Appendix Fig S19 for the images of more animals and time points.


Comparison of the body length , weight and external appearance of five wild - types and five mutants after the alternative treatments of two sets of antibiotic mixtures . The treatments were started with 8 - week - old fish and continued for six weeks . The measurements and photoshoots were conducted five weeks later . Data of n = 5 are represented in box ( min to max with mean line ) plots . Individual data points for each animal were plotted as grey dots . n . s . , no significant , * * P < 0 . 01 by student ' s t - test . The raw statistics was listed in Table EV3 .

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Comparison of the body length, weight and external appearance of five wild-types and five mutants after the alternative treatments of two sets of antibiotic mixtures. The treatments were started with 8-week-old fish and continued for six weeks. The measurements and photoshoots were conducted five weeks later. Data of n=5 are represented in box (min to max with mean line) plots. Individual data points for each animal were plotted as grey dots. n.s., no significant, ** P < 0.01 by student's t-test. The raw statistics was listed in Table EV3.


( C - D ) Snapshots from confocal live imaging movies of a representative embryo ( N = 7 ) during cellularisation co - expressing opto - Notch , Mito - Zdk , MCP : : GFP and sim - MS2 . Continuous photo - activation from the onset of cellularisation with a stack size of z = 25μm ( λ = 488 nm ) was alternated with mCherry excitation ( λ = 561 nm ) at 1 min intervals . Single confocal z - slices of the mCherry channel are shown to record opto - Notch localization before , and 10 minutes after sustained photo - activation . Magnified insets show opto - Notch bound to the mitochondrial anchor in the dark ( C ) and in the nucleus upon photo - activation ( D ) . Scale bar , 10 μm .

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(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.


( F - H ) Maximum intensity z - projections ( z = 25 μm ) of the same embryo simultaneously photoactivated and imaged in the GFP channel ( λ = 488 nm ) to record sim expression at a time resolution of 30 s . Shown are the first cycle of photo - activation , which approximates the dark state ( F ) , 15 min ( G ) , and 35 min ( H ) after continuous photo - activation . Magnified insets show that sim is not expressed in the dark at this stage ( F ) , but it is expressed in lateral ectodermal cells after 15 min of photo - activation ( G ) , and appeared to be shut off in most nuclei after 35 mins ( H ) . Scale bar , 10 μm .

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(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.


The number of sim - expressing nuclei as counted by automated spot detection over time ( A ) Continuous opto - Notch activation initially induces sim expression across a large number of nuclei . Over time , the majority of nuclei adapt to the input and terminate expression .

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The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression.


( D - E ) Quantification of nuclear localisation of mCherry : : NICD during opto - Notch photo - activation . Raw data was bleaching - corrected with an exponential fit and shown is the nuclear / total ratio , which further normalises bleaching and embryo - to - embryo variability . NICD displays persistent nuclear enrichment during continuous activation ( D ) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol ( E ) .

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(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).


( D - E ) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant ( Hairless H2 ) and under overexpression of Twist ( Twist OE ) The observed outcomes are most consistent with the predictions from the state - dependent inactivation model .

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(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE) The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.


( D ) Absolute cell numbers of the indicated cell types were determined by flow - cytometric analysis of the bone marrow from 3 - week - old Pax5 + / + and Pax5Jak2 / + mice . Average cell numbers are shown with SEM and were statistically analyzed by multiple t - tests ( unpaired two - tailed with Holm - Šídák ' s correction ) ; ns , not significant ( P > 0 . 05 ) . One of 3 independent experiments is shown .

(D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown.


( F ) Kaplan - Meier survival analysis of Pax5Jak2 / + ( black ) and control Pax5 + / + ( grey ) mice . n , number of mice analyzed . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test .

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(F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test.


( H ) Eosin - hematoxylin - stained sections of the lung and liver of a Pax5Jak2 / + tumor mouse . Infiltrating and blasting tumor cells are indicated by an arrow .

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(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.


( I ) Flow - cytometric analysis of lymph node cells from a control Pax5 + / + mouse and a 10 - week - old Pax5Jak2 / + tumor mouse . ( J ) Flow - cytometric analysis of B220lowCD19 + B cells from the bone marrow of a 4 - week - old Pax5Jak2 / + mouse .

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(I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.


( D - G ) GSEA analysis of 327 repressed ( D ) or 330 activated ( F ) Pax5 target genes identified in pro - B cells as compared with the ranked log2 - fold gene expression changes in Pax5Jak2 / + ( PJ ) B - ALLs versus control ( Ctrl ) Pax5 + / - Cdkn2ab + / - B - ALLs ( left ) . NES , normalized enrichment score . The expression of five genes that are upregulated in Pax5Jak2 / + B - ALL cells ( upper row ) and Pax5 - / - ( KO ) pro - B cells ( lower row ) is shown in ( E ) . Likewise , the expression of five genes that are upregulated in control B - ALL cells ( upper row ) and Pax5 + / + ( WT ) pro - B cells ( lower row ) is shown in ( G ) . TPM , transcripts per million . Mean TPM values with SEM are shown for the following RNA - seq experiments : 3 ( WT pro - B ) , 2 ( KO pro - B ) , 2 ( Ctrl B - ALL ) , and 4 ( PJ B - ALL ) .

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(D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL).


( H ) Expression of the Ptprc ( CD45 ) gene from exon 3 to exon 8 , as determined by RNA - seq of Pax5Jak2 / + and Pax5 + / - Cdkn2ab + / - B - ALL cells . The alternatively spliced exons 4 ( A ) , 5 ( B ) and 6 ( C ) are indicated in orange in the respective exon - intron structure of the Ptprc gene . Ptprc transcripts of B220 + Pax5 + / - Cdkn2ab + / - B - ALL cells contain all three exons , thus giving rise to expression of the CD45 isoform RABC ( known as B220 ) . In contrast , reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2 / + B - ALL cells , which likely gives rise to the CD45 isoforms RBC and

(H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB


( D ) Flow - cytometric analysis of bone marrow cells from 4 - week - old Pax5ihCd2 / + and Pax5Jak2 / ihCd2 mice . The expression of human ( h ) CD2 from the Pax5ihCd2 allele is shown for CD19 + B220 + ( grey ) and CD19 + B220low ( black ) B cells .

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(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.


( G ) Kaplan - Meier survival analysis of Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + ( black ) and Cd79a - Cre Pax5LSL - Jak2 / + ( grey ) mice . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; * * * * P < 0 . 0001 . n , number of mice analyzed .

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(G) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.


( H ) Flow - cytometric analysis of B220 and CD19 expression in B - ALL tumor cells from the lymph node of a Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + mouse ( black ; left ) . Pax5 expression in these B - ALL tumor cells ( black line ) and control Pax5 + / + lymph node B cells ( grey filled ) was determined by intracellular Pax5 staining ( right ) .

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(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).


( B , Flow - cytometric analysis of bone marrow and spleen from Pax5Jak2 - KD / + ( black ; n = 11 ) and control Pax5 + / + ( grey ; n = 7 ) mice at the age of 6 - 8 weeks . The frequencies of the indicated B cell types are shown for each organ ( B ) . The data are presented as mean percentages with SEM and were statistically analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) ; ns ( P > 0 . 5 ) , * P < 0 . 05 , * * P < 0 . 01 .

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(B, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01.


( D , Tumor progression in mice , transplanted with Pax5Jak2 - Luc / + tumor cells , in the presence or absence of the JAK1 / 2 inhibitor ruxolitinib . At day 14 after cell transfer , the transplanted mice were treated twice daily with ruxolitinib or vehicle , and the tumor mass was monitored by bioluminescence measurements . Images of three representative mice with or without ruxolitinib treatment are

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(D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown