C Quantification of peripheral ER structures in WT and ∆ ice2 cells harboring the inducible system ( SSY1405 , 1603 ) and treated with 800 nM estradiol for the times indicated . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of peripheral ER structures in WT and ∆ice2 cells harboring the inducible system (SSY1405, 1603) and treated with 800 nM estradiol for the times indicated. Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


E Quantification of peripheral ER structures in untreated WT , Δice2 , Δopi1 and Δice2 Δopi1 cells ( SSY1404 , 2356 , 2595 , 2811 ) . Bars are the mean percentage of cell cortex covered by tubules ( purple ) or sheets ( green ) , n = 3 biological replicates . Upper error bars are s . e . m . for the sum of tubules and sheets , lower error bars are s . e . m . for sheets . Asterisks indicate statistical significance compared with the corresponding value in WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 ; n . s . , not significant .

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

E Quantification of peripheral ER structures in untreated WT, Δice2, Δopi1 and Δice2 Δopi1 cells (SSY1404, 2356, 2595, 2811). Bars are the mean percentage of cell cortex covered by tubules (purple) or sheets (green), n = 3 biological replicates. Upper error bars are s.e.m. for the sum of tubules and sheets, lower error bars are s.e.m. for sheets. Asterisks indicate statistical significance compared with the corresponding value in WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01; n.s., not significant.


C , D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63 - mNeon and Rtn1 - mCherry ( SSY1405 , 1603 ) that were untreated ( C ) or treated with 8 mM DTT for 1 h ( D ) .

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

C, D Fluorescence images of cortical sections of WT and Δice2 cells expressing Sec63-mNeon and Rtn1-mCherry (SSY1405, 1603) that were untreated (C) or treated with 8 mM DTT for 1 h (D).


B Growth assays of untreated WT , Δice2 , Δnem1 , Δnem1 Δice2 , Δspo7 , Δspo7 Δice2 , Δpah1 and Δpah1 Δice2 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 , 2807 , 2808 ) . Numbers represent areas under the curves and serve as growth indices . Mean + s . e . m . , n = 3 biological replicates . Data for WT and ∆ ice2 cells are the same in the left and middle panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Growth assays of untreated WT, Δice2, Δnem1, Δnem1 Δice2, Δspo7, Δspo7 Δice2, Δpah1 and Δpah1 Δice2 cells (SSY1404, 2356, 2482, 2484, 2481, 2483, 2807, 2808). Numbers represent areas under the curves and serve as growth indices. Mean + s.e.m., n = 3 biological replicates. Data for WT and ∆ice2 cells are the same in the left and middle panels.


C , D Lipidomic analysis of WT , Δice2 , Δnem1 , Δice2 Δnem1 , Δspo7 and Δice2 Δspo7 cells ( SSY1404 , 2356 , 2482 , 2484 , 2481 , 2483 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * , P < 0 . 05 ; * * , P < 0 . 01 . Data for WT and Δice2 cells are the same as in both panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lipidomic analysis of WT, Δice2, Δnem1, Δice2 Δnem1, Δspo7 and Δice2 Δspo7 cells (SSY1404, 2356, 2482, 2484, 2481, 2483). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. *, P < 0.05; **, P < 0.01. Data for WT and Δice2 cells are the same as in both panels.


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

E Sec63-mNeon images of untreated WT, Δnem1, Δnem1Δice2, Δspo7 and Δspo7 Δice2 cells (SSY1404, 2482, 2484, 2481, 2483).


D Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1 - HA from ∆ nem1 mutants ( SSY3065 ) and microsomes from cells of the indicated genotypes ( SSY3053 , 3074 , 3075 , 3095 ) . Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase ( PPase ) are shown for reference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot of HA from Pah1 dephosphorylation reactions that contained phosphorylated Pah1-HA from ∆nem1 mutants (SSY3065) and microsomes from cells of the indicated genotypes (SSY3053, 3074, 3075, 3095). Phosphorylated Pah1 and dephosphorylated Pah1 resulting from treatment with recombinant alkaline phosphatase (PPase) are shown for reference.


E Western blot of HA , Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ ice2 cells expressing Nem1 - HA ( SSY3140 , 3141 ) . Nem1 is undetectable in cell lysates due to its low abundance .

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

E Western blot of HA, Sec61 and Pgk1 from cell lysates and microsomes prepared from WT and ∆ice2 cells expressing Nem1-HA (SSY3140, 3141). Nem1 is undetectable in cell lysates due to its low abundance.


Western blots of FLAG , HA and Dpm1 from cell lysates or anti - FLAG immunoprecipitates of WT or ∆ nem1 cells containing Spo7 - FLAG , Nem1 - FLAG or Ice2 - HA as indicated ( SSY122 , 2421 , 3183 , 3184 , 3195 , 3196 , 3197 ) . The asterisk marks the position of the light chain from the antibody used for immunoprecipitation . The diamond marks a protein that is non - specifically precipitated by the anti - FLAG antibody . IP , immunoprecipitate .

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

Western blots of FLAG, HA and Dpm1 from cell lysates or anti-FLAG immunoprecipitates of WT or ∆nem1 cells containing Spo7-FLAG, Nem1-FLAG or Ice2-HA as indicated (SSY122, 2421, 3183, 3184, 3195, 3196, 3197). The asterisk marks the position of the light chain from the antibody used for immunoprecipitation. The diamond marks a protein that is non-specifically precipitated by the anti-FLAG antibody. IP, immunoprecipitate.


E Images of cells with endogenously tagged Ice2 - mScarlet and Spo7 - mNeon or Nem1 - mNeon ( SSY3244 , 3245 ) . Arrows indicate foci containg both Ice2 and either Spo7 or Nem1 . F Quantification of Spo7 and Nem1 foci in WT and ∆ ice2 cells ( SSY2916 , 3238 , 2917 , 3239 ) . Mean + s . e . m . , n = 3 biological replicates . Asterisks indicate statistical significance compared with the respective WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 .

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

E Images of cells with endogenously tagged Ice2-mScarlet and Spo7-mNeon or Nem1-mNeon (SSY3244, 3245). Arrows indicate foci containg both Ice2 and either Spo7 or Nem1. F Quantification of Spo7 and Nem1 foci in WT and ∆ice2 cells (SSY2916, 3238, 2917, 3239). Mean + s.e.m., n = 3 biological replicates. Asterisks indicate statistical significance compared with the respective WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


A Lipidomic analysis of WT and ∆ ice2 cells in which PAH1 was replaced with PAH1 - HA or pah1 ( 7A ) - HA as indicated ( SSY2841 , SSY2842 , SSY2970 ) . Mean + s . e . m . , n = 4 biological replicates . Asterisks indicate statistical significance compared with WT cells , as judged by a two - tailed Student ' s t - test assuming equal variance . * * , P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lipidomic analysis of WT and ∆ice2 cells in which PAH1 was replaced with PAH1-HA or pah1(7A)-HA as indicated (SSY2841, SSY2842, SSY2970). Mean + s.e.m., n = 4 biological replicates. Asterisks indicate statistical significance compared with WT cells, as judged by a two-tailed Student's t-test assuming equal variance. **, P < 0.01.


A Sec63 - mNeon images of mid and cortical sections of untreated WT and ∆ opi1 cells , overexpressing ICE2 where indicated ( SSY1404 , 2588 , 2595 , 2596 ) .

O O O O O O O O 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 Sec63-mNeon images of mid and cortical sections of untreated WT and ∆opi1 cells, overexpressing ICE2 where indicated (SSY1404, 2588, 2595, 2596).


D Growth assays on solid media of WT , ∆ hac1 , Δice2 , and Δhac1 Δice2 cells ( SSY1404 , 2356 , 2805 , 2806 ) in the absence or presence of 0 . 2 µg / ml tunicamycin . For each series , cells were diluted fivefold from one step to the next .

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

D Growth assays on solid media of WT, ∆hac1, Δice2, and Δhac1 Δice2 cells (SSY1404, 2356, 2805, 2806) in the absence or presence of 0.2 µg/ml tunicamycin. For each series, cells were diluted fivefold from one step to the next.


A . Example of littermate males of the indicated genotypes 26 days post partum . Additional examples are provided in Appendix Fig S2 .

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. Example of littermate males of the indicated genotypes 26 days post partum. Additional examples are provided in Appendix Fig S2.


B . Weights of mice of the indicated genotypes from 18 to 120 days post partum . Boxplots indicate 5 - 95 percentile . A minimum of 8 animals is plotted at each age . Asterisks denote statistical significance ( NS , not significant , * P value < 0 . 05 , * * P value < 0 . 01 and * * * P value < 0 . 001 ) determined by the unpaired two - way Wilcoxon rank - sum test .

B. Weights of mice of the indicated genotypes from 18 to 120 days post partum. Boxplots indicate 5-95 percentile. A minimum of 8 animals is plotted at each age. Asterisks denote statistical significance (NS, not significant, *P value <0.05, **P value <0.01 and ***P value <0.001) determined by the unpaired two-way Wilcoxon rank-sum test.


C . Example of dissected brains ( upper panel ) and coronal sections ( left and right panels ) stained with H & amp ; amp ; E of the indicated genotypes . Note the severe hydrocephaly and ventricular dilation of the lateral ventricle ( LV ) and third ventricle ( TV ) in a Gemc1 - / - mouse ( bottom right panel ) compared to a Gemc1 + / + littermate animal ( bottom left panel ) . Periventricular malacia ( asterisks ) and hippocampal atrophy ( arrow ) is also evident in the Gemc1 - / - sample . Scale bars = 500 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

C. Example of dissected brains (upper panel) and coronal sections (left and right panels) stained with H&amp;E of the indicated genotypes. Note the severe hydrocephaly and ventricular dilation of the lateral ventricle (LV) and third ventricle (TV) in a Gemc1-/- mouse (bottom right panel) compared to a Gemc1+/+ littermate animal (bottom left panel). Periventricular malacia (asterisks) and hippocampal atrophy (arrow) is also evident in the Gemc1-/- sample. Scale bars = 500 µm.


D . Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers ( n ) . The study was terminated at 16 months and mice were examined for histology . 97 % ( 31 / 32 ) of the Gemc1 - / - mice examined to date exhibited hydrocephaly ( additional histological examples in Appendix Fig S2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kaplan Meyer survival curve of a cohort of mice of the indicated genotypes and numbers (n). The study was terminated at 16 months and mice were examined for histology. 97% (31/32) of the Gemc1-/- mice examined to date exhibited hydrocephaly (additional histological examples in Appendix Fig S2).


E . RT - QPCR analysis of Gemc1 expression in murine tissues from Gemc1 + / + mice using a Taqman probe . Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted .

E. RT-QPCR analysis of Gemc1 expression in murine tissues from Gemc1+/+ mice using a Taqman probe. Data is compiled from duplicate samples of 3 male mice per tissue and mean and standard deviation are plotted.


F . RT - QPCR analysis of Gemc1 in trachea , oviduct and ovary tissue from Gemc1 + / + and Gemc1 - / - mice shows that the knockout reduces Gemc1 mRNA to undetectable levels . Mean and standard deviation of duplicate samples from 2 female mice are plotted . Additional examples from other Gemc1 - / - tissues are shown in Appendix Fig S1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-QPCR analysis of Gemc1 in trachea, oviduct and ovary tissue from Gemc1+/+ and Gemc1-/- mice shows that the knockout reduces Gemc1 mRNA to undetectable levels. Mean and standard deviation of duplicate samples from 2 female mice are plotted. Additional examples from other Gemc1-/- tissues are shown in Appendix Fig S1.


. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age .

. Example of dissected testes from littermate mice of the indicated genotypes at 2 months of age.


B . Testes from Gemc1 - / - mice have strongly reduced cellularity compared to Gemc1 + / + or Gemc1 + / - animals , as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber ( n = 3 ) . Mean and standard deviation are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Testes from Gemc1-/- mice have strongly reduced cellularity compared to Gemc1+/+ or Gemc1+/- animals, as measured by disaggregation of the tissue and cell counting with a Neubauer Chamber (n=3). Mean and standard deviation are indicated.


C . Histological analysis of tubules from 6 - week - old Gemc1 - / - mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids . Scale bars = 100 µm ( left panels ) and 50 µm ( right panels ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Histological analysis of tubules from 6-week-old Gemc1-/- mice revealed thinning of the spermatogenic cell layer and decreased numbers of elongated spermatids. Scale bars = 100 µm (left panels) and 50 µm (right panels).


D . Sperm counts from the cauda epididymis of wild type and Gemc1 - / - mice revealed no sperm in mice lacking GEMC1 ( n = 3 ) . Mean and standard deviation are indicated . E . Examples of sperm morphology from spermatocyte spreads from 2 month old mice . Scale bars = 10 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

D. Sperm counts from the cauda epididymis of wild type and Gemc1-/- mice revealed no sperm in mice lacking GEMC1 (n=3). Mean and standard deviation are indicated. E. Examples of sperm morphology from spermatocyte spreads from 2 month old mice. Scale bars = 10 µm.


F and G . Histological evaluation of the caput epididymis in sagittal ( F ) and coronal ( G ) orientation from 2 and 4 month old animals , respectively . Note the absence of luminal spermatozoa in the Gemc1 - / - mice compared to wild type . Scale bars = 100 µm ( F , left panels ) and 50 µm ( F , right panels and G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G. Histological evaluation of the caput epididymis in sagittal (F) and coronal (G) orientation from 2 and 4 month old animals, respectively. Note the absence of luminal spermatozoa in the Gemc1-/- mice compared to wild type. Scale bars = 100 µm (F, left panels) and 50 µm (F, right panels and G).


A . Examples of ovaries from Gemc1 + / + ( top panels ) and Gemc1 - / - mice ( bottom panels ) at 6 weeks of age . Ovaries were smaller in Gemc1 - / - mice and contained primarily degenerated antral follicles . A histologically normal antral follicle is indicated in the Gemc1 + / + ovary ( top right , black arrow ) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1 - / - ovaries ( bottom right , red arrows ) . Scale bars = 200 µm ( left panels ) ) and 100 µm ( right top panels and right bottom panel respectively ) . B . Morphological abnormalities in the oviduct of 3 - week - old Gemc1 - / - mice ( right panels ) compared to Gemc1 + / + ( left panels ) . A clear lack of cilia ( bottom panels ) in Gemc1 - / - oviducts is observed by H & amp ; amp ; E staining . Scale bars = 50 µm ( top panels ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Examples of ovaries from Gemc1+/+ (top panels) and Gemc1-/- mice (bottom panels) at 6 weeks of age. Ovaries were smaller in Gemc1-/- mice and contained primarily degenerated antral follicles. A histologically normal antral follicle is indicated in the Gemc1+/+ ovary (top right, black arrow) and degenerated antral follicles with high levels of cell death are indicated in the Gemc1-/- ovaries (bottom right, red arrows). Scale bars = 200 µm (left panels)) and 100 µm (right top panels and right bottom panel respectively). B. Morphological abnormalities in the oviduct of 3-week-old Gemc1-/- mice (right panels) compared to Gemc1+/+ (left panels). A clear lack of cilia (bottom panels) in Gemc1-/- oviducts is observed by H&amp;E staining. Scale bars = 50 µm (top panels).


C . Staining of frozen sections of oviduct with antibodies for Ac - tubulin ( cilia ) and γ - tubulin ( basal bodies ) reveals normal cilia organization in the wild type oviduct ( left ) while basal bodies and cilia are not detectable in Gemc1 - / - oviducts ( right ) . D . FOXJ1 and E . RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1 + / + oviducts and absent in Gemc1 - / - mice . Scale bars = 50 µm ( C - E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Staining of frozen sections of oviduct with antibodies for Ac-tubulin (cilia) and γ-tubulin (basal bodies) reveals normal cilia organization in the wild type oviduct (left) while basal bodies and cilia are not detectable in Gemc1-/- oviducts (right). D. FOXJ1 and E. RFX3 staining that is characteristic of MCCs was clearly evident in Gemc1+/+ oviducts and absent in Gemc1-/- mice. Scale bars = 50 µm (C-E).


A . H & amp ; amp ; E staining of the nasal mucosa of E17 . 5 embryos of the indicated genotype . Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1 + / + embryos compared to the attenuated , flat columnar to squamous epithelium of Gemc1 - / - embryos . Scale bars = 20 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

A. H&amp;E staining of the nasal mucosa of E17.5 embryos of the indicated genotype. Normal respiratory pseudostratified columnar ciliated epithelium is shown in Gemc1+/+ embryos compared to the attenuated, flat columnar to squamous epithelium of Gemc1-/- embryos. Scale bars = 20 µm.


B . PAS staining ( purple ) of the nasal mucosa of E18 . 5 embryos of the indicated genotype . Normal PAS positive goblet cells ( inset , red arrows ) are interspersed between MCCs ( inset , black arrows ) with their characteristic goblet or cup - like shape in Gemc1 + / + embryos . The Gemc1 - / - mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell ( inset , red arrows ) and lacks typical goblet cells . Scale bars = 20 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

B. PAS staining (purple) of the nasal mucosa of E18.5 embryos of the indicated genotype. Normal PAS positive goblet cells (inset, red arrows) are interspersed between MCCs (inset, black arrows) with their characteristic goblet or cup-like shape in Gemc1+/+ embryos. The Gemc1-/- mucosa has abundant PAS positive materials that accumulate in the apical borders of almost every columnar epithelial cell (inset, red arrows) and lacks typical goblet cells. Scale bars = 20 µm.


C . Transmission electron micrographs of cross - sections of adult trachea from Gemc1 + / + animals revealed the presence of secretory cells ( SC ) and multiciliated cells ( MCC ) . The apical membrane of MCCs accumulated basal bodies ( BB ) nucleating multiple cilia ( C ) and microvilli ( MV ) . In the Gemc1 - / - airway epithelium , no MCCs are identifiable and only secretory - like cells with MV at the apical surface are detectable . Scale bars = 5µm . Higher magnifications of the boxed regions are shown below , scale bars = 0 . 5µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transmission electron micrographs of cross-sections of adult trachea from Gemc1+/+ animals revealed the presence of secretory cells (SC) and multiciliated cells (MCC). The apical membrane of MCCs accumulated basal bodies (BB) nucleating multiple cilia (C) and microvilli (MV). In the Gemc1-/- airway epithelium, no MCCs are identifiable and only secretory-like cells with MV at the apical surface are detectable. Scale bars = 5µm. Higher magnifications of the boxed regions are shown below, scale bars = 0.5µm.


D . Lack of Ac - tubulin ( top panel ) and RFX3 ( second panel from top ) staining in the trachea of 1 month old mice .

O O O O O 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. Lack of Ac-tubulin (top panel) and RFX3 (second panel from top) staining in the trachea of 1 month old mice.


E . Cilia ( Ac - tubulin staining ) are not detectable in the ependymal cells of 2 month old Gemc1 - / - mice ( right panel ) . Choroid plexus ( CP ) and lateral ventricles ( LV ) are indicated . Scale bars = 20

E. Cilia (Ac-tubulin staining) are not detectable in the ependymal cells of 2 month old Gemc1-/- mice (right panel). Choroid plexus (CP) and lateral ventricles (LV) are indicated. Scale bars = 20 µm


E and F . RT - QPCR analysis of individual genes related to MCC formation in the oviduct ( E ) and trachea ( F ) of Gemc1 - / - mice compared to wild type littermates , using 18S rRNA as a reference ( n = 2 ) . Genes examined generally fall into 3 classes : centriole duplication ( Cep152 , Plk4 , Cetn2 ) , Deuterosome mediated centriole expansion ( Deup1 , Ccdc78 , Ccno ) and transcription of genes required for MCC differentiation ( FoxJ1 , Myb , Mcidas ) .

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

E and F. RT-QPCR analysis of individual genes related to MCC formation in the oviduct (E) and trachea (F) of Gemc1-/- mice compared to wild type littermates, using 18S rRNA as a reference (n=2). Genes examined generally fall into 3 classes: centriole duplication (Cep152, Plk4, Cetn2), Deuterosome mediated centriole expansion (Deup1, Ccdc78, Ccno) and transcription of genes required for MCC differentiation (FoxJ1, Myb, Mcidas).


A . GEMC1 interacts with E2F4 and DP1 . Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel . FLAGimmunoprecipiation ( IP ) of GEMC1co - IPs DP1 ( α - HA , lane 5 ) and E2F4 ( α - HA , lane 6 ) . The interaction is enhanced upon co - expression of HA tagged E2F4 and DP1 ( α - HA , lane 7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GEMC1 interacts with E2F4 and DP1. Western blot of HA or FLAG tagged protein expression in the input lysates are shown in the top panel. FLAGimmunoprecipiation (IP) of GEMC1co-IPs DP1 (α-HA, lane 5) and E2F4 (α- HA, lane 6). The interaction is enhanced upon co-expression of HA tagged E2F4 and DP1 (α-HA, lane 7).


B . GEMC1 interacts with E2F5 but not E2F1 . Co - IP of E2F5 ( α - HA , lane 3 ) and E2F5 - DP1 ( α - HA , lane 6 ) but not E2F1 or E2F1 - DP1 ( α - HA , lanes 2 and 5 ) . In the FLAG - IP panels , a short ( top ) and long ( second from top ) exposure of the Western are shown .

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B. GEMC1 interacts with E2F5 but not E2F1. Co-IP of E2F5 (α-HA, lane 3) and E2F5-DP1 (α-HA, lane 6) but not E2F1 or E2F1-DP1 (α-HA, lanes 2 and 5). In the FLAG-IP panels, a short (top) and long (second from top) exposure of the Western are shown.


C . Identification of the domain required for E2F4 interaction . GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP . Wild type GEMC1 ( lane 5 ) or GEMC1 lacking the CC domain ( lane 6 ) pulled down E2F4 - DP1 . A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4 - DP1 ( lanes 7 , 8 ) .

C. Identification of the domain required for E2F4 interaction. GFP tagged GEMC1 and mutant forms were expressed and IPs were performed against GFP. Wild type GEMC1 (lane 5) or GEMC1 lacking the CC domain (lane 6) pulled down E2F4-DP1. A GEMC1 protein lacking the TIRT domain or GFP alone did not IP E2F4-DP1 (lanes 7, 8).


D . Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients ( G313D ) impairs the interaction with E2F4 - DP1 ( compare land 5 and 6 ) .

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D. Mutation of the single amino acid analogous that identified in MCIDAS in human RGMC patients (G313D) impairs the interaction with E2F4-DP1 (compare land 5 and 6).


E . GEMC1 interacts with Multicilin through its CC domain . Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc - tagged Multicilin ( anti - Myc blot , lanes 4 and 6 ) while a CC domain mutant does not ( anti - Myc blot , lane 5 ) .

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E. GEMC1 interacts with Multicilin through its CC domain. Wild type GEMC1 or a mutant lacking the TIRT domain pull down Myc-tagged Multicilin (anti- Myc blot, lanes 4 and 6) while a CC domain mutant does not (anti-Myc blot, lane 5).


A . Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression ( RT - QPCR ) in HEK293T cells . Results for MCIDAS are graphed on a smaller scale in the right panel : V = vector and G = GEMC1 . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar induction of FOXJ1 was observed in U2OS and T98G ( Fig EV3 ) .

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A. Transient overexpression of GEMC1 leads to increased levels of FOXJ1 and MCIDAS expression (RT-QPCR) in HEK293T cells. Results for MCIDAS are graphed on a smaller scale in the right panel: V = vector and G = GEMC1. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar induction of FOXJ1 was observed in U2OS and T98G (Fig EV3).


B . Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 ( RT - QPCR ) . A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells . The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed . Similar results were also observed in U2OS ( Fig EV3 ) .

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B. Both the CC and the TIRT domain of GEMC1 are required to activate FOXJ1 (RT-QPCR). A corresponding Western blot showing relative expression levels of the mutant GFP tagged GEMC1 variants is shown from HEK293T cells. The mean and standard deviation of 3 individual experiments in HEK293T cells are graphed. Similar results were also observed in U2OS (Fig EV3).


C . Activation of FOXJ1 by GEMC1 is inhibited by the co - expression of Geminin ( compare 3rd and 4th lanes ) . Example Western blot is shown below . Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells .

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C. Activation of FOXJ1 by GEMC1 is inhibited by the co-expression of Geminin (compare 3rd and 4th lanes). Example Western blot is shown below. Results graphed are the mean and standard deviation of 3 individual experiments in HEK293T cells.


D . Co - expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression ( measured by RT - QPCR ) as Multicilin alone in HEK293T cells . The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed .

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D. Co-expression of GEMC1 and Multicilin results in similar levels of FOXJ1 expression (measured by RT-QPCR) as Multicilin alone in HEK293T cells. The mean and standard deviation of 4 individual experiments in HEK293T cells are graphed.


E and F . The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin . HEK293T cells were transfected with a control siRNA ( siC ) or 2 different siRNAs against MCIDAS ( si1 and si2 ) prior to being transfected with GFP or GFP - GEMC1 expression vectors . Relative MCIDAS ( E ) and FOXJ1 ( F ) expression levels were assessed by RT - QPCR . The mean and standard deviation of a representative experiment performed in triplicate is graphed . An additional experiment is presented in Fig EV3 .

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E and F. The activation of FOXJ1 by GEMC1 is partially dependent on Multicilin. HEK293T cells were transfected with a control siRNA (siC) or 2 different siRNAs against MCIDAS (si1 and si2) prior to being transfected with GFP or GFP-GEMC1 expression vectors. Relative MCIDAS (E) and FOXJ1 (F) expression levels were assessed by RT-QPCR. The mean and standard deviation of a representative experiment performed in triplicate is graphed. An additional experiment is presented in Fig EV3.


A ) Transcriptome comparison of hemocytes from stage 16 embryos ( E16 ) and wandering 3rd instar larvae ( WL ) . The x - axis is the average genes expression levels ( n = 3 ) and the y - axis the Log2 Fold Change WL / E16 . P - values are indicated with the color code .

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A) Transcriptome comparison of hemocytes from stage 16 embryos (E16) and wandering 3rd instar larvae (WL). The x-axis is the average genes expression levels (n=3) and the y-axis the Log2 Fold Change WL/E16. P-values are indicated with the color code.


Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in hemocytes

Scatter plots as in (A) highlighting in black subsets of known genes expressed in hemocytes


Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in


Scatter plots as in ( A ) highlighting in black subsets of known genes associated with the GO terms extracellular matrix

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Scatter plots as in (A) highlighting in black subsets of known genes associated with the GO terms extracellular matrix


Scatter plots as in ( A ) highlighting in black subsets of known genes associated with the GO terms extracellular


Scatter plots as in ( A ) highlighting in black subsets of known genes expressed in phagocytosis

Scatter plots as in (A) highlighting in black subsets of known genes expressed in phagocytosis


F - G ) Phagocytosis assay on E16 ( F ) and WL hemocytes ( G ) srp ( hemo ) - moesin - RFP . The beads ( in green ) are phagocytosed by the hemocytes ( in red ) . The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure . Full stacks are displayed and the scale bars represent 20 µm .

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F-G) Phagocytosis assay on E16 (F) and WL hemocytes (G) srp(hemo)-moesin-RFP. The beads (in green) are phagocytosed by the hemocytes (in red). The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure. Full stacks are displayed and the scale bars represent 20 µm.


A ) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL ( NI dataset ) .

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A) UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL (NI dataset).


D ) Top 5 markers of each cluster . The expression levels is represented by the gradient of purple levels ) and the percentage of cells with the size of the dots .

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D) Top 5 markers of each cluster. The expression levels is represented by the gradient of purple levels) and the percentage of cells with the size of the dots.


A ) Identification of the position ( circulating / resident ) of the clusters within the larva by qPCR . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment . Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment ( n = 5 , mean + / - SD are represented on the graph ) . The p - values are estimated by bilateral paired student test

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A) Identification of the position (circulating/resident) of the clusters within the larva by qPCR. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment. Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment (n=5, mean +/- SD are represented on the graph). The p-values are estimated by bilateral paired student test


A ) Identification of the position ( circulating / resident ) of the clusters within the larva by qPCR . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the right panel indicates the Log2 of the ratio between the expression level in the circulating versus the resident compartment . Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment ( n = 5 , mean + / - SD are represented on the graph ) . The p - values are estimated by bilateral paired student

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B ) Circulating and resident hemocytes ( top and bottom panels , respectively ) from Mimic - Lsp1beta - MI05460 WL , which express Lsp1beta tagged with GFP . The immunolabelling was done with anti - GFP ( in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI and GFP , the right panels show the GFP alone . The scale bars represent 10 µm . C ) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC - Lsp1beta - MI05460 larvae . The OregonR value indicates the background level . The number of cells included in the analysis is displayed on the x - axis label and were quantified from two independent preparation , p - values were estimated after variance analysis with bilateral student test for equal variance .

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B) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes from MiMIC-Lsp1beta-MI05460 larvae. The OregonR value indicates the background level. The number of cells included in the analysis is displayed on the x-axis label and were quantified from two independent preparation, p-values were estimated after variance analysis with bilateral student test for equal variance.


B ) Circulating and resident hemocytes ( top and bottom panels , respectively ) from Mimic - Lsp1beta - MI05460 WL , which express Lsp1beta tagged with GFP . The immunolabelling was done with anti - GFP ( in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI and GFP , the right panels show the GFP alone . The scale bars represent 10 µm . C ) Quantification of the GFP intensity in circulating and resident

B) Circulating and resident hemocytes (top and bottom panels, respectively) from Mimic-Lsp1beta-MI05460 WL, which express Lsp1beta tagged with GFP. The immunolabelling was done with anti-GFP (in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI and GFP, the right panels show the GFP alone. The scale bars represent 10 µm. C) Quantification of the GFP intensity in circulating and resident hemocytes


D ) Resident hemocytes located around the oenocytes or along the muscles ( top and bottom panels , respectively ) from WL srp ( hemo ) - moesin - RFP , that express moesin tagged with RFP in hemocytes . The immunolabelling was done with anti - RFP ( in red ) , anti - Pendulin ( Pen , in gray ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , Pen and RFP , the middle panels RFP alone and the right panels Pen alone . Arrow heads in the top panels indicate cells co - expressing RFP and Pen . The scale bars represent 50 µm .

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D) Resident hemocytes located around the oenocytes or along the muscles (top and bottom panels, respectively) from WL srp(hemo)-moesin-RFP, that express moesin tagged with RFP in hemocytes. The immunolabelling was done with anti-RFP (in red), anti-Pendulin (Pen, in gray) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, Pen and RFP, the middle panels RFP alone and the right panels Pen alone. Arrow heads in the top panels indicate cells co-expressing RFP and Pen. The scale bars represent 50 µm.


A ) Heatmap representing the z - score for the top 5 regulons of each cluster determined with SCENIC . The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset . The z - score is indicated with a gradient from magenta ( z - score < 0 , the regulon is repressed ) to yellow ( z - score > 0 , the regulon is active ) .

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A) Heatmap representing the z-score for the top 5 regulons of each cluster determined with SCENIC. The dendrogram on the left side of the panel indicates the correlation between the regulons across the dataset. The z-score is indicated with a gradient from magenta (z-score < 0, the regulon is repressed) to yellow (z-score > 0, the regulon is active).


A ) UMAP projection representing the 16 clusters of cells identified in the WI sample . Note the presence of two additional clusters , LMLM - 1 and LM - 2 , compared to the NI sample . The two clusters correspond to the lamellocytes .

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A) UMAP projection representing the 16 clusters of cells identified in the WI sample. Note the presence of two additional clusters, LMLM-1 and LM-2, compared to the NI sample. The two clusters correspond to the lamellocytes.


B ) Estimation of the number of cells of each cluster per larva in normal condition ( NI , orange ) and after wasp infestation ( WI , blue ) . For each cluster , the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets .

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B) Estimation of the number of cells of each cluster per larva in normal condition (NI, orange) and after wasp infestation (WI, blue). For each cluster, the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets.


D ) Dot plot representing the expression levels ( gradient of red levels ) and the percentage of cells ( size of the dots ) that express the top 5 markers of each cluster in the WI dataset .

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D) Dot plot representing the expression levels (gradient of red levels) and the percentage of cells (size of the dots) that express the top 5 markers of each cluster in the WI dataset.


A ) Scatter plot comparing the transcriptome of the two lamellocyte clusters , deduced from the WI dataset . The x - axis is the average expression levels of the genes and the y - axis the log2 fold change ( LM - 1 / LM - 2 ) . The genes significantly enriched in the LM - 1 cluster are highlighted in red ( p < 10 - 6 ) , those significantly enriched in the LM - 2 cluster in orange ( p < 10 - 6 ) .

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A) Scatter plot comparing the transcriptome of the two lamellocyte clusters, deduced from the WI dataset. The x-axis is the average expression levels of the genes and the y-axis the log2 fold change (LM-1/LM-2). The genes significantly enriched in the LM-1 cluster are highlighted in red (p<10-6), those significantly enriched in the LM-2 cluster in orange (p<10-6).


C ) Expression levels ( gradient of color ) and the percentage of cells ( size of the dots ) for the top 10 markers of lamellocytes ( in black ) , of markers specific to LM - 1 compared to LM - 2 ( in red ) and specific to LM - 2 compared to LM - 1 ( in orange ) . Note that most markers enriched in LM - 1 compared to LM - 2 are exclusively expressed in LM - 1 while most markers enriched in LM - 2 compared to LM - 1 are also expressed in the plasmatocyte and crystal cell clusters .

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C) Expression levels (gradient of color) and the percentage of cells (size of the dots) for the top 10 markers of lamellocytes (in black), of markers specific to LM-1 compared to LM-2 (in red) and specific to LM-2 compared to LM-1 (in orange). Note that most markers enriched in LM-1 compared to LM-2 are exclusively expressed in LM-1 while most markers enriched in LM-2 compared to LM-1 are also expressed in the plasmatocyte and crystal cell clusters.


D ) Hemocytes from WL OregonR infested by wasp . The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea ( in gray , top panels ) and Shot ( in gray , lower panels ) . Phalloidin - FITC ( in green ) labels the actin filament particularly abundant in lamellocytes ( Tokusumi et al . , 2009 ) and the nuclei were marked with DAPI ( blue ) . Full stacks are displayed , the left panels show the overlay of DAPI , FITC and the lamellocyte markers , the middle panels show the FITC alone and the right panels the lamellocyte markers alone . The scale bars represent 20µm .

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D) Hemocytes from WL OregonR infested by wasp. The immunolabelling was done with antibodies targeting the new lamellocyte markers identified in this study Rhea (in gray, top panels) and Shot (in gray, lower panels). Phalloidin-FITC (in green) labels the actin filament particularly abundant in lamellocytes (Tokusumi et al., 2009) and the nuclei were marked with DAPI (blue). Full stacks are displayed, the left panels show the overlay of DAPI, FITC and the lamellocyte markers, the middle panels show the FITC alone and the right panels the lamellocyte markers alone. The scale bars represent 20µm.


A ) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae . The left panel indicates the distribution of each marker across all clusters ( as in Figure 2D ) , the graph on the right panel , indicates the Log2 of the ratio between the expression level in WI versus NI . Positive values indicate an up - regulation upon wasp infestation and negative values a down - regulation ( n = 3 , pool of 10 larvae per replicate , mean + / - SD are represented on the graph ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ .

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A) Expression levels of the cluster markers in the hemolymph of WI larvae compared to NI larvae. The left panel indicates the distribution of each marker across all clusters (as in Figure 2D), the graph on the right panel, indicates the Log2 of the ratio between the expression level in WI versus NI. Positive values indicate an up-regulation upon wasp infestation and negative values a down-regulation (n=3, pool of 10 larvae per replicate, mean +/- SD are represented on the graph). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[.


B , C ) Expression levels of the cluster markers increasing in WI compared to NI according to ( A ) during the progression of the immune response to wasp infestation . Collections were carried out at t0 ( time of infestation , L2 ) , 24 h ( early L3 ) , 48 h ( mid L3 ) and 72 h ( WL ) after wasp infestation ( n = 3 ) . The time line is on the x - axis and the normalised expression levels on the y - axis . The expression levels were normalised to the levels at t0 . The dotted lines indicate t0 relative level ( = 1 ) . The markers that do not display strong variability during the timeline are presented in ( B ) and the ones showing a strong up - regulation are in ( C ) . D ) Expression levels during the infestation timeline as in ( B , C ) for the markers of proliferation .

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B,C) Expression levels of the cluster markers increasing in WI compared to NI according to (A) during the progression of the immune response to wasp infestation. Collections were carried out at t0 (time of infestation, L2), 24 h (early L3), 48 h (mid L3) and 72 h (WL) after wasp infestation (n=3). The time line is on the x-axis and the normalised expression levels on the y-axis. The expression levels were normalised to the levels at t0. The dotted lines indicate t0 relative level (=1). The markers that do not display strong variability during the timeline are presented in (B) and the ones showing a strong up-regulation are in (C). D) Expression levels during the infestation timeline as in (B,C) for the markers of proliferation.


B , C ) Expression levels of the cluster markers increasing in WI compared to NI according to ( A ) during the progression of the immune response to wasp infestation . Collections were carried out at t0 ( time of infestation , L2 ) , 24 h ( early L3 ) , 48 h ( mid L3 ) and 72 h ( WL ) after wasp infestation ( n = 3 ) . The time line is on the x - axis and the normalised expression levels on the y - axis . The expression levels were normalised to the levels at t0 . The dotted lines indicate t0 relative level ( = 1 ) . The markers that do not display strong variability during the timeline are presented in ( B ) and the ones showing a strong up - regulation are in ( C ) .

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B,C) Expression levels of the cluster markers increasing in WI compared to NI according to (A) during the progression of the immune response to wasp infestation. Collections were carried out at t0 (time of infestation, L2), 24 h (early L3), 48 h (mid L3) and 72 h (WL) after wasp infestation (n=3). The time line is on the x-axis and the normalised expression levels on the y-axis. The expression levels were normalised to the levels at t0. The dotted lines indicate t0 relative level (=1). The markers that do not display strong variability during the timeline are presented in (B) and the ones showing a strong up-regulation are in (C).


E ) Weighted dot plot representing the enrichment ( Log2 fold change ) of the markers in hemocytes originating from the lymph gland ( DotGFP - WI ) compared to embryonic hemocytes ( srpGFP - WI ) from WI larva . The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp ( hemo ) Gal4 respectively combined with a lineage tracing construct . The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue ( not significant ) to light blue ( p < 10 - 3 ) . The p - value are estimated by bilateral student test and indicated as follow : ns = not - significant ( > 0 . 05 ) , " * " = p [ 0 . 05 - 0 . 01 [ , " * * " = p [ 0 . 01 - 0 . 001 [ , " * * * " = p < 0 . 001 ] .

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

E) Weighted dot plot representing the enrichment (Log2 fold change) of the markers in hemocytes originating from the lymph gland (DotGFP-WI) compared to embryonic hemocytes (srpGFP-WI) from WI larva. The lymph gland hemocytes and embryonic hemocytes were traced using the lymph gland driver DotGal4 and the embryonic driver Srp(hemo)Gal4 respectively combined with a lineage tracing construct. The relative expression levels are indicated by the size of the dots and the pvalue of the log2 fold change with the gradient dark blue (not significant) to light blue (p<10-3). The p-value are estimated by bilateral student test and indicated as follow: ns = not-significant (>0.05), "*" = p[0.05 - 0.01[, "**" = p[0.01-0.001[, "***" = p <0.001].


Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( A ) Induced Cyp1a1 mRNA expression in response to different treatments ( n = 3 per group ) .

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Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (A) Induced Cyp1a1 mRNA expression in response to different treatments (n=3 per group).


Colonic organoids grown from Lgr5 - EGFP - IRES - CreERT2 mice were treated with an AhR agonist ( 10 nM TCDD ) or antagonist ( 10 µM CH223191 ) for 3 d . ( B ) Assessment of GFP + stem cell percentages ( n = 3 replicates per treatment ) . Bars with different letters are significantly different ( p < 0 . 05 ) .

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Colonic organoids grown from Lgr5-EGFP-IRES-CreERT2 mice were treated with an AhR agonist (10 nM TCDD) or antagonist (10 µM CH223191) for 3 d. (B) Assessment of GFP+ stem cell percentages (n=3 replicates per treatment). Bars with different letters are significantly different (p<0.05).


( D ) Flow cytometry analysis of GFP + Tomato + and Tomato + cells in the large intestine of AhR wild type ( Lgr5 - GFPCreERT2 X Tomatof / f , GT ) and AhR knock out ( AhRf / f X Lgr5 - GFPCreERT2 X Tomatof / f , HGT ) mice . Top left panel shows representative cells from WT negative control animal ( no GFP or Tomato expression ) . Top right panel shows Tomato only control ( CDX2P - CreERT2 X Tomatof / f ) . Middle panels represent DMSO or TCDD treatment in AhR WT mice , respectively . Mice were gavaged every other day , 4 times total , with vehicle or TCDD ( 25 µg / kg bw ) and terminated 1 d later . Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse .

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

(D) Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type (Lgr5-GFPCreERT2 X Tomatof/f, GT) and AhR knock out (AhRf/f X Lgr5-GFPCreERT2 X Tomatof/f, HGT) mice. Top left panel shows representative cells from WT negative control animal (no GFP or Tomato expression). Top right panel shows Tomato only control (CDX2P-CreERT2 X Tomatof/f). Middle panels represent DMSO or TCDD treatment in AhR WT mice, respectively. Mice were gavaged every other day, 4 times total, with vehicle or TCDD (25 µg/kg bw) and terminated 1 d later. Bottom left panel is from a representative AhR WT mouse and bottom right panel is from a representative AhR KO mouse.


( E ) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD ( n = 4 ) .

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(E) Induced Cyp1a1 mRNA expression in stem cells after oral gavage with TCDD (n=4).


( F ) Percentage of Lgr5 + stem cells ( n = 4 mice per group ) in ( D ) .

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(F) Percentage of Lgr5+ stem cells (n=4 mice per group) in (D).


Flow cytometry analysis of GFP + Tomato + and Tomato + cells in the large intestine of AhR wild type and AhR knock out mice . ( G ) Quantitative analysis of the percentage of Lgr5 + stem cells in ( D ) , n = 7 - 10 mice per group .

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Flow cytometry analysis of GFP+Tomato+ and Tomato+ cells in the large intestine of AhR wild type and AhR knock out mice. (G) Quantitative analysis of the percentage of Lgr5+ stem cells in (D), n=7-10 mice per group.


Representative immunohistochemistry of GFP + stem cells per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm .

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Representative immunohistochemistry of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm.


Representative violin plots of GFP + stem cells per crypt , n = 60 ~ 70 crypts from n = 3 mice per group . Scale 50 µm . Solid line indicates median , and dotted lines indicate 25th / 75th quartiles .

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Representative violin plots of GFP+ stem cells per crypt, n=60~70 crypts from n=3 mice per group. Scale 50 µm. Solid line indicates median, and dotted lines indicate 25th/75th quartiles.


( A ) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5 - EGFP - IRES - CreERT2 mice .

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(A) Representative flow cytometry gating and fluorescence microscopy images of sorted GFPhigh stem cells and GFPlow progenitor cells from Lgr5-EGFP-IRES-CreERT2 mice.


( B ) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice ( Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ GC ] ) and KO mice ( AhRf / f X Lgr5 - GFPCreERT2 X CDX2P - CreERT2 [ HGC ] ) , treated with DMSO ( control ) or TCDD for 5 d . Data are from ≥ 4 mice , scale bar 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 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

(B) Representative brightfield images of organoids generated from GFPhigh stem cells isolated from tamoxifen treated AhR WT mice (Lgr5-GFPCreERT2 X CDX2P-CreERT2 [GC]) and KO mice (AhRf/f X Lgr5-GFPCreERT2 X CDX2P-CreERT2 [HGC]), treated with DMSO (control) or TCDD for 5 d. Data are from ≥4 mice, scale bar 200 µm.


( C , D ) The organoid forming efficiency ( C ) and diameter ( D ) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD ( 10 nM ) . n = 4 - 5 mice per group in ( C ) . Each dot represents an individual organoid and grey line indicates mean in ( D ) . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(C, D) The organoid forming efficiency (C) and diameter (D) from sorted GFPhigh stem cells in AhR WT or KO mice treated for 5 d with DMSO or TCDD (10 nM). n=4-5 mice per group in (C). Each dot represents an individual organoid and grey line indicates mean in (D). Bars with different letters are significantly different (p<0.05).


Organoid growth from GFPlow progenitor cells . ( E ) Representative brightfield images of organoids .

Organoid growth from GFPlow progenitor cells. (E) Representative brightfield images of organoids.


( F ) Organoid forming efficiency and ( G ) diameter . n = 4 - 8 mice per group in ( F ) . Each dot represents an individual organoid and grey line indicates mean in ( G ) . Scale bar 200 μm . Bars with different letters are significantly different ( p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Organoid forming efficiency and (G) diameter. n=4-8 mice per group in (F). Each dot represents an individual organoid and grey line indicates mean in (G). Scale bar 200 μm. Bars with different letters are significantly different (p<0.05).


( H , I ) ( H ) Lgr5 expression in organoids and ( I ) sorted stem cells in the absence or presence of TCDD treatment ( n ≥ 3 independent samples per group ) .

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(H, I) (H) Lgr5 expression in organoids and (I) sorted stem cells in the absence or presence of TCDD treatment (n≥3 independent samples per group).


( J ) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d . Scale 200 µm .

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(J) Representative brightfield images for secondary organoid formation pretreated with DMSO or 10 nM TCDD for 2d. Scale 200 µm.


( C ) Heatmap comparison of the top 25 upstream regulators between AhR KO ( KO / WT ) and TCDD ( TCDD / vehicle in WT mice ) treated cells .

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(C) Heatmap comparison of the top 25 upstream regulators between AhR KO (KO/WT) and TCDD (TCDD/vehicle in WT mice) treated cells.


( A ) FoxM1 , Plk1 , and Cdc25B mRNA expression in stem and progenitor cells with and without AhR , n = 4 mice per group .

(A) FoxM1, Plk1, and Cdc25B mRNA expression in stem and progenitor cells with and without AhR, n=4 mice per group.


( B ) FoxM1 , Plk1 , and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD , n = 3 - 4 mice per group .

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(B) FoxM1, Plk1, and Cdc25B mRNA expression in WT stem and progenitor cells with and without TCDD, n=3-4 mice per group.


Chromatin immunoprecipitation ( ChIP ) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD , n = 3 or 4 per group . A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control .

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Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT colonic organoids treated with DMSO or TCDD, n=3 or 4 per group. A DNA fragment amplified in a gene desert on mouse chromosome 6 was used as a negative control.


Chromatin immunoprecipitation ( ChIP ) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts ( E ) treated with DMSO or TCDD , n = 3 or 4 per group .

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Chromatin immunoprecipitation (ChIP) analysis of AhR and FoxM1 promoter interaction using WT and KO crypts (E) treated with DMSO or TCDD, n=3 or 4 per group.


( F ) FoxM1 luciferase activity determination for young adult mouse colonocytes ( YAMCs ) treated with DMSO or 10 nM TCDD for 1 d , n = 3 per group .

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(F) FoxM1 luciferase activity determination for young adult mouse colonocytes (YAMCs) treated with DMSO or 10 nM TCDD for 1 d, n=3 per group.


( G Expression of FoxM1 and PLK1 at mRNA level , , n > = 3 independent organoids per treatment .

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(G Expression of FoxM1 and PLK1 at mRNA level, , n>=3 independent organoids per treatment.


H ) Expression of FoxM1 and PLK1 at protein level , n > = 3 independent organoids per treatment .

H) Expression of FoxM1 and PLK1 at protein level, n>=3 independent organoids per treatment.


( I ) Representative brightfield images of mouse colonic organoids generated from WT Lgr5 + - GFPhi stem cells treated with DMSO or 5µM FDI - 6 . Scale bar 200 µm .

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(I) Representative brightfield images of mouse colonic organoids generated from WT Lgr5+-GFPhi stem cells treated with DMSO or 5µM FDI-6. Scale bar 200 µm.


( J , K ) Quantification of organoid forming efficiency ( J ) and organoid area ( K ) from WT colonic stem cells treated with DMSO or 5µM FDI - 6 . Data are represented as mean ± SEM in ( K ) .

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(J, K) Quantification of organoid forming efficiency (J) and organoid area (K) from WT colonic stem cells treated with DMSO or 5µM FDI-6. Data are represented as mean ± SEM in (K).


( L ) Organoid expression of Lgr5 in response to FDI - 6 . Lgr5 expression level was dramatically suppressed by FDI - 6 in a dose - dependent manner , n > = 3 per treatment .

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(L) Organoid expression of Lgr5 in response to FDI-6. Lgr5 expression level was dramatically suppressed by FDI-6 in a dose-dependent manner, n>=3 per treatment.


( M ) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI - 6 for 2 d . Scale 200 µm .

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(M) Representative brightfield images for secondary organoid formation following treatment with DMSO or 5 µM FDI-6 for 2 d. Scale 200 µm.


( A and B ) Proliferating Lgr5 + stem cells marked by EdU in the distal colon from AhR WT ( GT ) and KO ( HGT ) mice . Arrowhead and star symbols denote proliferating stem cells ( GFP + EdU + ) and static stem cells ( GFP + EdU - ) , respectively , during a 2 h EdU pulse , n = 3 per treatment .

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(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.


( C ) Total number of EdU + cells per crypt in the distal colon of AhR WT and KO ( HGT ) mice , n = 3 mice per treatment . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( D & E ) Crypt cell proliferative zone analysis in AhR WT and KO ( HGT ) mice , presented as the distance from the crypt base to the highest EdU + cell divided by total crypt length . Red lines between crosses show the measurement of distance . Each symbol represents one crypt , n = 3 mice per treatment , scale bar 20 µm . Grey line represents mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.


( F & AhR KO promoted cell proliferation 5 d post DSS treatment . AhR WT ( AhRf / f [ H ] , n = 4 ) and AhR KO ( AhRf / f X Villin - Cre [ HV ] , n = 4 ) mice were used .

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(F& AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used.


G ) AhR KO promoted cell proliferation 5 d post DSS treatment . Data represent mean ± SEM , quantified as the ratio of the EdU + area relative to the DAPI + area using KeyenceTM software .

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G) AhR KO promoted cell proliferation 5 d post DSS treatment. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.


( H ) Increased cell proliferation ( EdU + cells ) observed in AhR KO vs WT organoids derived from HGC and GC mice , respectively ( n = 6 from 3 mice per group ) .

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(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).


( I ) Effect of AhR KO on cell cycle S and G2 - M phases ( n = 3 mice per group ) .

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(I) Effect of AhR KO on cell cycle S and G2-M phases (n=3 mice per group).


( J ) . FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation . Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton ( 50 mg / kg ) for 2 consecutive days , n = 80 ~ 100 crypts from 4 mice per group . Scale bar , 50 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles . Bars with different letters are significantly different ( p < 0 . 05 ) .

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(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).


( B ) Representative colon images from AhR WT and AhR KO mice following AOM / DSS treatment . Scale bar , 9 mm .

(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.


( C ) H & E staining of representative colon tumor sections from AhR WT and KO mice , scale bar 200 μm .

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(C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm.


( E , F ) ( E ) Adenoma and ( F ) adenocarcinoma multiplicity in AhR WT and KO mice .

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(E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice.


( G ) Tumor volume in AhR KO and WT mice . Grey line indicates mean .

(G) Tumor volume in AhR KO and WT mice. Grey line indicates mean.


( H ) FoxM1 mRNA expression in normal ( uninvolved ) crypts adjacent to colonic tumors . Images were quantified and expressed as FoxM1 positive area per crypt ( n = 5 mice per group ) . Scale 200 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( I ) FoxM1 mRNA expression in colon tumors . FoxM1 mRNA ISH area was averaged per tumor on each slide ( n = 5 mice per group ) . Scale 200 µm . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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(I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( A - B ) mRNA expression of human colonic stem cell markers LGR5 and OLFM4 , n = 5 or 6 independent human samples . Paired student t - test .

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(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test.


( C ) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD . Scale bar 50 µm .

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(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.


mRNA expression of target genes , n = 5 or 6 independent human samples . Paired student t - test .

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mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.


( H ) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n = 6 independent samples . Total protein was used as a loading control .

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(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.


( I ) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD . n = 3 - 4 per group .

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(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.


( J ) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d . C56 and C61 designate different human subjects .

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(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.


( K ) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon . Subsequent sequencing revealed indels at the expected locations .

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(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.


( L ) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies ( n = 304 ) . AhR low ( n = 152 ) is defined as below its median expression ; AhR high ( n = 152 ) is defined as above its median expression . Dashed line indicates median and dotted lines indicate 25th / 75th quartiles .

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

(L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.


( M ) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies ( n = 303 ) analyzed by Spearman rank correlation .

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

(M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation.


( N ) Representative brightfield images of human colonic organoids from sorted live cells at day 6 , scale bar 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

(N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm.


( O , P ) ( O ) Human organoid forming efficiency , and ( P ) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO , TCDD and CH223191 ( 10 μM ) for 6 d . n = 5 per group , 3 independent experiments . Grey line indicates mean in ( P ) . Treatments not sharing a common letter are significantly different , p < 0 . 05 .

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

(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.


( Q ) Representative brightfield images of human colonic organoids treated with DMSO or FDI - 6 . FoxM1 inhibition suppressed human colonic organoid growth in a dose - dependent manner , scale bar 200 µm .

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

(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.


Volcano plots of piliation , aggregation and adhesion mutation scores for all pilE mutations . Stop and synonymous mutations are highlighted in blue and orange respectively . Adhesion and aggregation mutation scores relative to piliation . The correlation appears as a red dotted line .

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

Volcano plots of piliation, aggregation and adhesion mutation scores for all pilE mutations. Stop and synonymous mutations are highlighted in blue and orange respectively. Adhesion and aggregation mutation scores relative to piliation. The correlation appears as a red dotted line.


Scanning electron microscopy of single bacteria on a cellulose filter . Scale bars : 500 nm .

Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.


Two representative immunofluorescence labelling of TFP . Scale bar : 1 µm .

Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm.


Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody . Black dots indicate all the individual de novo generated PilE mutants that were characterized . Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region . Solid lines indicate 95 % confidence interval . Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants . Mean values ± SEM are indicated for each strain . N ≥ 3 independent experiments . Paired t - test . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) .

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

Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval. Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).


Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody .

Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.


Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated ( magenta ) . In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling . Scale bar : 2 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

Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.


Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported . Each dot represents values for a PilE point mutant relative to the SB strain . Hypo - aggregative mutants are highlighted in orange while hyper - aggregative mutants are in light blue . solid lines indicate the 95 % confidence interval .

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

Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval.


Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot . PilX levels were normalized using PilE as a reference and normalized to pilESB values . Mean ratio ± SEM is indicated . N = 3 independent experiments . Paired 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 O O O O O O O O O O O O

Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.


Early adhesion of pilE mutants as a function of aggregation . Values were normalized to that of pilESB . Each dot represents a pilE mutant and the pilV , pilD , pilX and pilC1 reference mutants . Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple . A blown - up view of the zone of interest ( lower left quarter ) is presented on the right panel . The shaded area highlights the absence of significant adhesion .

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

Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion.


Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB .

Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB.


Piliation length of a selection of mutants determined by immunofluorescence . Average pilus length ± SEM is indicated . N = 6 experiments with at least 50 bacteria analyzed in each experiment . Paired t - test . Number of pili per bacteria ± SEM determined as in ( E ) . Paired t - test . p < 0 . 05 ( * ) .

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

Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test. Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*).


Time - lapse of a cell - adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes . Initiation of flow corresponds to t = 0 . Scale bar : 1 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

Time-lapse of a cell-adhered pilus fluorescently labeled with the F10 nanobody at three different focal planes. Initiation of flow corresponds to t=0. Scale bar: 1 µm.


Spinning - disk confocal immunofluorescence of pili stained with both SM1 ( magenta ) and F10 ( green ) . The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light . In each field of view , a substrate - and a cell - bound pilus are shown . Upper panels : z - projection from the stacks in the xy plane ; bottom panels : side view in the z plane . Scale bar : 2 µm . White arrows correspond to SM1 staining at the tip of pili . Quantification of adhesion after infection of HUVEC cells for 30 minutes , expressed as the number of adherent bacteria over the number of cell nuclei . Prior to infection , bacteria were incubated at 37 ° C with or without antibody or nanobody for 15 minutes . Mean values ± SEM are indicated for each strain . N = 5 independent experiments . Paired t - test . p < 0 . 05 ( * ) .

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

Spinning-disk confocal immunofluorescence of pili stained with both SM1 (magenta) and F10 (green). The thick appearance of TFP is due to the primary and secondary reagents used to detect them as well as diffraction of light. In each field of view, a substrate- and a cell-bound pilus are shown. Upper panels: z-projection from the stacks in the xy plane; bottom panels: side view in the z plane. Scale bar: 2 µm. White arrows correspond to SM1 staining at the tip of pili. Quantification of adhesion after infection of HUVEC cells for 30 minutes, expressed as the number of adherent bacteria over the number of cell nuclei. Prior to infection, bacteria were incubated at 37°C with or without antibody or nanobody for 15 minutes. Mean values ± SEM are indicated for each strain. N=5 independent experiments. Paired t-test. p<0.05 (*).


Isolated sporozoites from the hemolymph ( HL ) and salivary glands ( SG ) of infected mosquitoes stained with Hoechst and SiR - tubulin . Note the similar staining of WT and α1 - tubulin ( - ) complemented lines . Scale bars : 5 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

Isolated sporozoites from the hemolymph (HL) and salivary glands (SG) of infected mosquitoes stained with Hoechst and SiR-tubulin. Note the similar staining of WT and α1-tubulin(-) complemented lines. Scale bars: 5 µm.


SiR - tubulin staining ( green , I and II ) and TEM ( III ) show that microtubules shrink after wild type sporozoite formation . During budding microtubules can be up to 15 µm long ( I ) , while after formation they measure on average only 6 µm ( graph ) . Inset in II shows sporozoite cross - section at the sporoblast membrane ; note the SiR - tubulin fluorescence ( green , arrowheads ) next to the nuclear Hoechst stain ( blue ) . ( III ) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule ( white line , length ) . Green arrowheads point to microtubules close to the sporoblast . Rh : rhoptry ; N : nucleus ; ER : endoplasmic reticulum ; Mi : mitochondrion ; Sb : sporoblast . * * * * indicate p < 0 . 0001 ; Kruskal - Wallis - test . Scale bars : I & II : 5 µm , magnification box in II : 1 µm , III : 1 µm , magnification box in III : 0 . 1 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

SiR-tubulin staining (green, I and II) and TEM (III) show that microtubules shrink after wild type sporozoite formation. During budding microtubules can be up to 15 µm long (I), while after formation they measure on average only 6 µm (graph). Inset in II shows sporozoite cross-section at the sporoblast membrane; note the SiR-tubulin fluorescence (green, arrowheads) next to the nuclear Hoechst stain (blue). (III) TEM longitudinal section of budding sporozoites with an indicated subpellicular microtubule (white line, length). Green arrowheads point to microtubules close to the sporoblast. Rh: rhoptry; N: nucleus; ER: endoplasmic reticulum; Mi: mitochondrion; Sb: sporoblast. **** indicate p<0.0001; Kruskal-Wallis-test. Scale bars: I&II: 5 µm, magnification box in II: 1 µm, III: 1 µm, magnification box in III: 0.1 µm.


Comparison of sporozoite development in oocysts of WT , α1 - tubulin ( - ) and complemented lines . Hoechst ( blue ) and SiR - tubulin ( green ) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls , while SiR tubulin staining is absent from α1 - tubulin ( - ) oocysts . mCherry stains the cytoplasm of the parasite . Scale bars : 5 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

Comparison of sporozoite development in oocysts of WT, α1-tubulin(-) and complemented lines. Hoechst (blue) and SiR-tubulin (green) staining reveal that the complemented line generates sporozoites indistinguishable from WT controls, while SiR tubulin staining is absent from α1-tubulin(-) oocysts. mCherry stains the cytoplasm of the parasite. Scale bars: 5 µm.


Transmission electron micrographs of developing sporozoites in WT and α1 - tubulin ( - ) parasite lines . Note the slender sporozoites in the WT oocysts , which are absent in the mutant . Sb : sporoblast ; scale bars : 5 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

Transmission electron micrographs of developing sporozoites in WT and α1-tubulin(-) parasite lines. Note the slender sporozoites in the WT oocysts, which are absent in the mutant. Sb: sporoblast; scale bars: 5 µm.


TEM cross sections of WT and α1 - tubulin ( - ) parasites showing the aberrant structure of the latter . Subpellicular and centriolar plaque ( spindle pole body ) associated ( hemispindle ) microtubules are readily recognizable in the WT sporozoites ( green arrowheads in insert ) but absent in the mutant , while the inner membrane complex is formed below the plasma membrane of both parasites ( white arrowheads ) . Note the different scale chosen to highlight the malformed shapes in the mutant . N : nucleus ; scale bars : 0 . 2 µm , magnification boxes : 0 . 1 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

TEM cross sections of WT and α1-tubulin(-) parasites showing the aberrant structure of the latter. Subpellicular and centriolar plaque (spindle pole body) associated (hemispindle) microtubules are readily recognizable in the WT sporozoites (green arrowheads in insert) but absent in the mutant, while the inner membrane complex is formed below the plasma membrane of both parasites (white arrowheads). Note the different scale chosen to highlight the malformed shapes in the mutant. N: nucleus; scale bars: 0.2 µm, magnification boxes: 0.1 µm,.


Electron micrographs showing different stages of development in WT and α1 - tubulin ( - ) parasites . Key structural elements are highlighted in the following colors : blue : plasma membrane ; yellow : inner membrane complex ; green : microtubules ; light blue : micronemes ; lilac : rhoptries ; brown : rootlet fiber ; cyan : nucleus . Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1 - tubulin ( - ) parasites . The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque ( dark structure in nuclear envelope ) . Panel III corresponds to Fig 1D . Scale 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

Electron micrographs showing different stages of development in WT and α1-tubulin(-) parasites. Key structural elements are highlighted in the following colors: blue: plasma membrane; yellow: inner membrane complex; green: microtubules; light blue: micronemes; lilac: rhoptries; brown: rootlet fiber; cyan: nucleus. Note the undulating shapes of the elongated mutants and that micronemes are hard to distinguish from other small vesicles in the α1-tubulin(-) parasites. The rootlet fiber in panel II starts at the apical tip and in panel V is associated with the centriolar plaque (dark structure in nuclear envelope). Panel III corresponds to Fig 1D. Scale bars: 200 nm.


α1 - tubulin ( - ) parasites show multiple attachments ( arrowheads ) with the sporoblast membrane at day 17 post infection . Note the highly branched nature of the budding parasite ( same parasite as in panel C ) shown in two views in I and II . Right : slices through the tomogram in grey scale with the outlines used for the 3D model indicated . Two connections with the sporoblast are indicated by 1 and 2 . Scale bars : 1 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

α1-tubulin(-) parasites show multiple attachments (arrowheads) with the sporoblast membrane at day 17 post infection. Note the highly branched nature of the budding parasite (same parasite as in panel C) shown in two views in I and II. Right: slices through the tomogram in grey scale with the outlines used for the 3D model indicated. Two connections with the sporoblast are indicated by 1 and 2. Scale bars: 1 µm.