f2 ( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult animals expressing HLH - 30 : : GFP . Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 ° C ( three biological replicates , ~ 50 animals each , mean ± s . d . , * * P0 . 01 , Student ' s t - test ) . b , c ) Expression of putative autophagy - related and lysosomal target genes was measured by quantitative PCR in day 1 ( b ) WT ( N2 ) and ( c ) hlh - 30 ( tm1978 ) animals fed bacteria expressing control or tor dsRNA from hatching ( 20 ° C ) . Data are mean ± s . d . of biological triplicates . * P0 . 05 , * * P0 . 01 ; Student ' s t - test . csta * is a cathepsin A orthologue ( cosmid C08H9 . 1 ( ref . 30 ) ) . ( d ) Lifespan analysis of WT animals and hlh - 30 ( tm1978 ) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 ° C . See Supplementary Table S3 for details of lifespan analyses and replicate experiments .

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

f2(a) Nuclear localization of HLH-30 was quantified in day 1 adult animals expressing HLH-30::GFP. Animals were fed bacteria expressing control or tor dsRNA from hatching and raised at 20 °C (three biological replicates, ~50 animals each, mean±s.d., **P0.01, Student's t-test).b,c) Expression of putative autophagy-related and lysosomal target genes was measured by quantitative PCR in day 1 (b) WT (N2) and (c) hlh-30(tm1978) animals fed bacteria expressing control or tor dsRNA from hatching (20 °C). Data are mean±s.d. of biological triplicates. *P0.05, **P0.01; Student's t-test. csta* is a cathepsin A orthologue (cosmid C08H9.1 (ref. 30)).(d) Lifespan analysis of WT animals and hlh-30(tm1978) mutants fed bacteria expressing control or tor dsRNA from day 1 of adulthood was carried out at 20 °C. See Supplementary Table S3 for details of lifespan analyses and replicate experiments.


f3Lifespan analyses of ( a ) WT ( N2 ) and ( b ) germline - less glp - 1 ( e2141 ) animals raised at the non - permissive temperature ( 25 ° C ) and fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood were carried out at 20 ° C . Lifespan analyses of ( c ) dietary - restricted eat - 2 ( ad1116 ) mutants , ( d ) insulin / IGF - 1 receptor daf - 2 ( e1370 ) mutants , ( e ) mitochondrial respiration clk - 1 ( e2519 ) mutants and ( f ) mRNA translation rsks - 1 ( sv31 ) mutants , fed bacteria expressing control or hlh - 30 dsRNA from day 1 of adulthood ( c , d , f ) or larval L4 stage ( e ) , were carried out at 20 ° C . See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments .

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

f3Lifespan analyses of (a) WT (N2) and (b) germline-less glp-1(e2141) animals raised at the non-permissive temperature (25 °C) and fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood were carried out at 20 °C. Lifespan analyses of (c) dietary-restricted eat-2(ad1116) mutants, (d) insulin/IGF-1 receptor daf-2(e1370) mutants, (e) mitochondrial respiration clk-1(e2519) mutants and (f) mRNA translation rsks-1(sv31) mutants, fed bacteria expressing control or hlh-30 dsRNA from day 1 of adulthood (c,d,f) or larval L4 stage (e), were carried out at 20 °C. See Supplementary Table S2 for details of lifespan analyses including at least three independent experiments.


f4 ( a ) Nuclear localization of HLH - 30 was quantified in day 1 adult WT , eat - 2 , daf - 2 , clk - 1 and rsks - 1 mutants ( mean ± s . d . of four biological replicates , ~ 50 animals each , * * P0 . 01 , analysis of variance ) . ( b ) Expression of hlh - 30 was measured by quantitative PCR ( qPCR ) in day 1 adult WT ( N2 ) , eat - 2 ( ad1116 ) ( dietary restriction , DR ) , daf - 2 ( e1370 ) ( insulin / IGF - 1 signaling ) , clk - 1 ( e2519 ) ( mitochondrial respiration ) and rsks - 1 ( sv31 ) ( mRNA translation ) animals ( mean ± s . d . of three biological replicates , * P0 . 05 , Student ' s t - test ) . ( c ) Lifespan analysis of WT and transgenic animals overexpressing HLH - 30 : : GFP , and raised and maintained on OP50 was carried out at 20 ° C . See Supplementary Table S7 for details of lifespan analyses and replicate experiments . ( d ) GFP : : LGG - 1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH - 30 ( n > 300 , ± s . e . m . , * * P0 . 01 , Student ' s t - test ) . The increase in GFP : : LGG - 1 punctae observed in animals overexpressing HLH - 30 could be reversed by atg - 18 RNAi treatment ( data not shown ) . ( e ) TFEB expression was measured by qPCR in the livers of 4 . 5 - month - old female ( F ) and male ( M ) mice fed AL or subjected to DR for 5 . 5 weeks starting at 3 months of age ( mean ± s . e . m . of ~ 20 mice per group , * P0 . 05 , Student ' s t - test ) . ( f ) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR . Actin was included as a loading control , see Supplementary Fig . S8 for additional controls . ( g ) TFEB protein levels in Fig . 4f were quantified by densitometry and normalized to actin ( mean ± s . d . , * P0 . 05 , Student ' s t - test ) .

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

f4(a) Nuclear localization of HLH-30 was quantified in day 1 adult WT, eat-2, daf-2, clk-1 and rsks-1 mutants (mean±s.d. of four biological replicates, ~50 animals each, **P0.01, analysis of variance).(b) Expression of hlh-30 was measured by quantitative PCR (qPCR) in day 1 adult WT (N2), eat-2(ad1116) (dietary restriction, DR), daf-2(e1370) (insulin/IGF-1 signaling), clk-1(e2519) (mitochondrial respiration) and rsks-1(sv31) (mRNA translation) animals (mean±s.d. of three biological replicates, *P0.05, Student's t-test).(c) Lifespan analysis of WT and transgenic animals overexpressing HLH-30::GFP, and raised and maintained on OP50 was carried out at 20 °C. See Supplementary Table S7 for details of lifespan analyses and replicate experiments.(d) GFP::LGG-1 punctae were quantified in hypodermal seam cells of WT animals or animals overexpressing HLH-30 (n>300, ±s.e.m., **P0.01, Student's t-test). The increase in GFP::LGG-1 punctae observed in animals overexpressing HLH-30 could be reversed by atg-18 RNAi treatment (data not shown).(e) TFEB expression was measured by qPCR in the livers of 4.5-month-old female (F) and male (M) mice fed AL or subjected to DR for 5.5 weeks starting at 3 months of age (mean±s.e.m. of ~20 mice per group, *P0.05, Student's t-test).(f) TFEB protein was detected by western blotting of nuclear fractions from the livers of five female and five male mice fed AL or subjected to DR. Actin was included as a loading control, see Supplementary Fig. S8 for additional controls. (g) TFEB protein levels in Fig. 4f were quantified by densitometry and normalized to actin (mean±s.d., *P0.05, Student's t-test).


Figure 1B ) SUMO2 immunoprecipitation using crosslinked SUMO2 antibody beads and liver samples of wild - type C57BL6 / J mice in the fasted ( 16 h ) or re - fed ( 16 h fasted and re - fed 2 h ) states . Eluates analyzed by immunoblotting using anti - Prox1 antibodies and ponceau staining was used as loading control . Data information Data : mean ± SEM . Significance was determined by one - way ANOVA with Dunnett ´ s multiple comparison test relative to samples collected at ZT 12 . * * * P ≤ 0 . 001 . C ) 8 weeks old C57BL / 6N male mice were fasted ( food removed at ZT 12 ) or re - fed ( fasted from ZT4 to ZT12 for synchronization ; food re - introduced at ZT 12 ) . Tissue samples were collected at ZT 12 , 15 , 18 and 21 ( n = 4 ) . Liver lysates analyzed by immunoblotting using anti - Prox1 and anti - P _ S6K ( Thr389 ) antibodies ; β actin was detected as an input control . The quantification of SUMOylated Prox1 ( % ) and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown . qPCR data is presented as relative fold change normalized to the housekeeping gene TBP . Data information : Every dot represents one individual mouse . Data : mean ± SEM . Significance was determined by one - way ANOVA with Dunnett ´ s multiple comparison test relative to samples collected at ZT 12 . * * * P ≤ 0 . 001 .

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

Figure 1B) SUMO2 immunoprecipitation using crosslinked SUMO2 antibody beads and liver samples of wild-type C57BL6/J mice in the fasted (16 h) or re-fed (16 h fasted and re-fed 2 h) states. Eluates analyzed by immunoblotting using anti-Prox1 antibodies and ponceau staining was used as loading control. Data information Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.C) 8 weeks old C57BL/6N male mice were fasted (food removed at ZT 12) or re-fed (fasted from ZT4 to ZT12 for synchronization; food re-introduced at ZT 12). Tissue samples were collected at ZT 12, 15, 18 and 21 (n=4). Liver lysates analyzed by immunoblotting using anti-Prox1 and anti-P_S6K(Thr389) antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 (%) and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown. qPCR data is presented as relative fold change normalized to the housekeeping gene TBP. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by one-way ANOVA with Dunnett´s multiple comparison test relative to samples collected at ZT 12. *** P≤0.001.


Figure 2B ) HepG2 cells overexpressing HA - tagged mouse wild - type Prox1 ( wt ) , Prox1 K556R mutant or Prox1 E558A mutant . Cell lysates analyzed by immunoblotting using anti - HA antibodies ; tubulin was detected as an input control . C ) Mouse un - tagged Prox1 ( mProx1 ) was purified from HEK293T cells . Purified mProx1 was incubated with recombinant E1 and E2 enzyme together with either SUMO1 or SUMO2 . The enzymatic reactions were started with ATP and incubated for 15 , 30 and 60 min . A reaction with the Prox1 K556R mutant ( KR ) was used as a control . The 0 min sample was incubated without ATP . Samples were analyzed by immunoblotting using anti - Prox1 antibodies .

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

Figure 2B) HepG2 cells overexpressing HA-tagged mouse wild-type Prox1 (wt), Prox1 K556R mutant or Prox1 E558A mutant. Cell lysates analyzed by immunoblotting using anti-HA antibodies; tubulin was detected as an input control.C) Mouse un-tagged Prox1 (mProx1) was purified from HEK293T cells. Purified mProx1 was incubated with recombinant E1 and E2 enzyme together with either SUMO1 or SUMO2. The enzymatic reactions were started with ATP and incubated for 15, 30 and 60 min. A reaction with the Prox1 K556R mutant (KR) was used as a control. The 0 min sample was incubated without ATP. Samples were analyzed by immunoblotting using anti-Prox1 antibodies.


Figure 3A ) 6 weeks old C57BL / 6N male mice were fed either a 0 % cholesterol diet ( ctrl ) or a 2 % high cholesterol diet ( chol 2 % ) for 6 weeks . B ) 6 weeks old C57BL / 6N male mice were fed either a standard chow diet or a 60 % high fat diet ( HFD ) for 8 weeks . C ) 6 weeks old C57BL / 6N male mice were fed either a standard chow diet or a 45 % high fat + 20 % w / v fructose diet ( HF / hfD ) for 12 weeks . ( A - C ) Liver samples were collected at ZT 15 in the fasted ( 11 h ) and re - fed ( fasted 8h and re - fed 3 h ) states ( n = 4 - 5 ) . Liver lysates were analyzed by immunoblotting using anti - Prox1 antibodies ; β actin was detected as an input control . The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown . Data information : Every dot represents one individual mouse . Data : mean ± SEM . Significance was determined by two - way ANOVA with Sidak ´ s multiple comparison test between different groups and conditions . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

Figure 3A) 6 weeks old C57BL/6N male mice were fed either a 0% cholesterol diet (ctrl) or a 2 % high cholesterol diet (chol 2%) for 6 weeks. B) 6 weeks old C57BL/6N male mice were fed either a standard chow diet or a 60 % high fat diet (HFD) for 8 weeks. C) 6 weeks old C57BL/6N male mice were fed either a standard chow diet or a 45% high fat + 20% w/v fructose diet (HF/hfD) for 12 weeks. (A-C) Liver samples were collected at ZT 15 in the fasted (11 h) and re-fed (fasted 8h and re-fed 3 h) states (n=4-5). Liver lysates were analyzed by immunoblotting using anti-Prox1 antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. *P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001.


Figure 4B ) 8 weeks old Prox1K556R K . I . ( f / f ) male mice were injected with a control AAV ( Ctrl _ AAV ) , with an AAV to overexpress Cre recombinase ( Cre _ AAV ) or with phosphate - buffered saline ( PBS ) ( n = 4 ) . Liver tissue samples were collected 3 weeks later from mice fed ad libitum . Liver lysates were analyzed by immunoblotting using anti - Prox1 and anti - Cre antibodies ; Vcp was detected as an input control . The quantification of SUMOylated Prox1 and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown . qPCR data presented as relative fold change normalized to the housekeeping gene TBP . Data information : ( B ) Every dot represents one individual mouse . Data : mean ± SEM . Significance was determined by one - way ANOVA with Tukey ´ s multiple comparison test between groups . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

Figure 4B) 8 weeks old Prox1K556R K.I. (f/f) male mice were injected with a control AAV (Ctrl_AAV), with an AAV to overexpress Cre recombinase (Cre_AAV) or with phosphate-buffered saline (PBS) (n=4). Liver tissue samples were collected 3 weeks later from mice fed ad libitum. Liver lysates were analyzed by immunoblotting using anti-Prox1 and anti-Cre antibodies; Vcp was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression as well as Prox1 mRNA levels analyzed by qPCR are shown. qPCR data presented as relative fold change normalized to the housekeeping gene TBP. Data information: (B) Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by one-way ANOVA with Tukey´s multiple comparison test between groups. *** P≤0.001, **** P≤0.0001.


Figure 5A ) 8 weeks old Prox1K556R K . I . ( f / f ) male mice were injected with a control AAV ( Ctrl _ AAV ) or with an AAV to overexpress Cre recombinase ( Cre _ AAV ) and placed on a high - fat ( 45 % ) high - fructose ( 20 % w / v ) diet for 18 weeks . Liver samples were collected between ZT 16 and 17 in the fasted ( 8 h ) and re - fed ( fasted 8 h and re - fed 4 - 5 h ) states ( n = 7 - 8 ) . Liver lysates were analyzed by immunoblotting using anti - Prox1 and anti - Cre antibodies ; β actin was detected as an input control . The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown . Data information Every dot represents one individual mouse . Data : mean ± SEM . Significance was determined by two - way ANOVA with Sidak ´ s multiple comparison test between different groups and conditions . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 . B ) Serum analysis using a colorimetric - based serum analyzer . Levels of total cholesterol , high density lipoprotein ( HDL ) and low density lipoprotein ( LDL ) are shown . Data information : Every dot represents one individual mouse . Data : mean ± SEM . Significance was determined by two - way ANOVA with Sidak ´ s multiple comparison test between different groups and conditions . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 . C ) Equal amounts of serum were pooled and separated by FPLC , serum lipoprotein cholesterol profiles are shown .

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

Figure 5A) 8 weeks old Prox1K556R K.I. (f/f) male mice were injected with a control AAV (Ctrl_AAV) or with an AAV to overexpress Cre recombinase (Cre_AAV) and placed on a high-fat (45%) high-fructose (20% w/v) diet for 18 weeks. Liver samples were collected between ZT 16 and 17 in the fasted (8 h) and re-fed (fasted 8 h and re-fed 4-5 h) states (n=7-8). Liver lysates were analyzed by immunoblotting using anti-Prox1 and anti-Cre antibodies; β actin was detected as an input control. The quantification of SUMOylated Prox1 and total Prox1 protein expression are shown. Data information Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. * P≤0.05, ** P≤0.01, **** P≤0.0001.B) Serum analysis using a colorimetric-based serum analyzer. Levels of total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL) are shown. Data information: Every dot represents one individual mouse. Data: mean ±SEM. Significance was determined by two-way ANOVA with Sidak´s multiple comparison test between different groups and conditions. * P≤0.05, ** P≤0.01, **** P≤0.0001.C) Equal amounts of serum were pooled and separated by FPLC, serum lipoprotein cholesterol profiles are shown.


Figure 6C ) Heatmap displaying the expression of enriched genes involved in phase I , II and III of bile acid detoxification and bile acid synthesis in the liver of obese mice expressing the Prox1 K556R mutant ( Cre _ AAV ) in comparison to obese mice expressing wild - type Prox1 ( Ctrl _ AAV ) in the fasted state ( n = 4 ) . The occupation scores according to a published Chip - sequencing analysis on Prox1 Cyp7a1 was used as a reference gene . D ) Prox1 / LRH - 1 co - immunoprecipitation in HEK293A cells overexpressing HA - tagged Prox1 wild - type ( wt ) or K556R mutant ( KR ) and Myc - tagged LRH - 1 .

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

Figure 6C) Heatmap displaying the expression of enriched genes involved in phase I, II and III of bile acid detoxification and bile acid synthesis in the liver of obese mice expressing the Prox1 K556R mutant (Cre_AAV) in comparison to obese mice expressing wild-type Prox1 (Ctrl_AAV) in the fasted state (n=4). The occupation scores according to a published Chip- sequencing analysis on Prox1 Cyp7a1 was used as a reference gene.D) Prox1/LRH-1 co-immunoprecipitation in HEK293A cells overexpressing HA-tagged Prox1 wild-type (wt) or K556R mutant (KR) and Myc-tagged LRH-1.


Figure 1B . Whole exome sequencing was carried out and analysis revealed a missense p . L165F variant ( c . 493 C > T ) in the ALX1 homeodomain , heterozygous in the parents ( ALX1165L / 165F ) , wildtype in the unaffected sibling ( ALX1165L / 165L ) , and homozygous in both affected subjects ( ALX1165F / 165F ) . E . Immunofluorescence staining for pluripotent markers SSEA4 , OCT4 , SOX2 , and TRA - 1 - 60 and alkaline phosphatase staining of iPSC clones . One representative iPSC clone is shown for each genotype . Scale bar : 400 µm . F . Expression of pluripotent ( OCT4 , NANOG ) , endoderm ( Endo . , AFP , GATA4 , FOXA2 ) , ectoderm ( Ecto . , NESTIN , GFAP , SOX1 ) , and mesoderm ( Meso . , BRACH . ( BRACHYURY ) , RUNX1 , CD34 ) gene markers for ALX1165L / 165L ( green ) , ALX1165L / 165F ( red ) , and ALX1165F / 165F ( blue ) iPSC relative to undifferentiated cells ( UND ) . Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype . Significance : p = 0 . 0167 for OCT4 , p = 0 . 0005 for NANOG , p = 0 . 000004 for AFP , p = 0 . 0082 for GATA4 , p = 0 . 0137 for FOXA2 , p = 0 . 00002 for NESTIN , p = 0 . 0167 for GFAP , p = 0 . 0014 for SOX1 , p = 0 . 0117 for BRACHYURY , p = 0 . 0008 for RUNX1 and p = 0 . 0068 for CD34 when comparing undifferentiated and differentiated ALX1165L / 165L iPSC . p = 0 . 0013 for OCT4 , p = 0 . 0011 for NANOG , p = 0 . 0000003 for AFP , p = 0 . 0003 for GATA4 , p = 0 . 0063 for FOXA2 , p = 0 . 0001 for NESTIN , p = 0 . 027 for GFAP , p = 0 . 000002 for SOX1 , p = 0 . 000009 for BRACHYURY , p = 3e - 9 for RUNX1 and p = 0 . 000006 for CD34 when comparing undifferentiated and differentiated ALX1165F / 165L iPSC . p = 0 . 0201 for OCT4 , p = 0 . 006 for NANOG , p = 1x10 - 12 for AFP , p = 5x10 - 13 for GATA4 , p = 0 . 0031 for FOXA2 , p = 0 . 0292 for NESTIN , p = 0 . 00001 for GFAP , p = 6x10 - 7 for SOX1 , p = 0 . 0204 for BRACHYURY , p = 0 . 0009 for RUNX1 and p = 0 . 000003 for CD34 when comparing undifferentiated and differentiated ALX1165F / 165F iPSC . Data from each clone were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant .

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

Figure 1B. Whole exome sequencing was carried out and analysis revealed a missense p.L165F variant (c.493 C>T) in the ALX1 homeodomain, heterozygous in the parents (ALX1165L/165F), wildtype in the unaffected sibling (ALX1165L/165L), and homozygous in both affected subjects (ALX1165F/165F).E. Immunofluorescence staining for pluripotent markers SSEA4, OCT4, SOX2, and TRA-1-60 and alkaline phosphatase staining of iPSC clones. One representative iPSC clone is shown for each genotype. Scale bar: 400 µm.F. Expression of pluripotent (OCT4, NANOG), endoderm (Endo., AFP, GATA4, FOXA2), ectoderm (Ecto., NESTIN, GFAP, SOX1), and mesoderm (Meso., BRACH. (BRACHYURY), RUNX1, CD34) gene markers for ALX1165L/165L (green), ALX1165L/165F(red), and ALX1165F/165F (blue) iPSC relative to undifferentiated cells (UND). Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype. Significance: p=0.0167 for OCT4, p=0.0005 for NANOG, p= 0.000004 for AFP, p=0.0082 for GATA4, p=0.0137 for FOXA2, p=0.00002 for NESTIN, p=0.0167 for GFAP, p=0.0014 for SOX1, p=0.0117 for BRACHYURY, p=0.0008 for RUNX1 and p=0.0068 for CD34 when comparing undifferentiated and differentiated ALX1165L/165L iPSC. p=0.0013 for OCT4, p=0.0011 for NANOG, p=0.0000003 for AFP, p=0.0003 for GATA4, p=0.0063 for FOXA2, p=0.0001 for NESTIN, p=0.027 for GFAP, p =0.000002 for SOX1, p=0.000009 for BRACHYURY, p=3e-9 for RUNX1 and p=0.000006 for CD34 when comparing undifferentiated and differentiated ALX1165F/165L iPSC. p=0.0201 for OCT4, p=0.006 for NANOG, p=1x10-12 for AFP, p=5x10-13 for GATA4, p=0.0031 for FOXA2, p=0.0292 for NESTIN, p=0.00001 for GFAP, p=6x10-7 for SOX1, p=0.0204 for BRACHYURY, p=0.0009 for RUNX1 and p=0.000003 for CD34 when comparing undifferentiated and differentiated ALX1165F/165F iPSC. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.


Figure 2B . Images of iPSC and iPSC - derived NCC at Days 0 , 14 , and passage 4 following differentiation . Scale bars : 400 µm ( Day 0 ) , 200µm ( Day 14 , passage 4 ) .

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

Figure 2B. Images of iPSC and iPSC-derived NCC at Days 0, 14, and passage 4 following differentiation. Scale bars: 400 µm (Day 0), 200µm (Day 14, passage 4).


Figure 3Gene expression analysis across NCC differentiation of unaffected control ALX1165L / 165L ( green ) , heterozygous ALX1165L / 165F ( magenta ) and homozygous ALX1165F / 165F iPSC : ALX1 , neural plate border specifier genes ZIC1 , PAX7 , PAX3 , MSX1 , MSX2 , DLX5 ; neural crest specifier genes FOXD3 , P75 , TFAP2A , SNAI2 , TWIST1 ; and lineage specifier gene HAND2 . The RT - qPCR relative expression values were normalized to RPLP0 and GAPDH expression . Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype . Exact p - values are provided in Table EV1 . Data from each clone were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant .

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

Figure 3Gene expression analysis across NCC differentiation of unaffected control ALX1165L/165L (green), heterozygous ALX1165L/165F (magenta) and homozygous ALX1165F/165F iPSC: ALX1, neural plate border specifier genes ZIC1, PAX7, PAX3, MSX1, MSX2, DLX5; neural crest specifier genes FOXD3, P75, TFAP2A, SNAI2, TWIST1; and lineage specifier gene HAND2. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. Exact p-values are provided in Table EV1. Data from each clone were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.


Figure 4A . Homozygous ALX1165F / 165F NCC ( blue ) showed an increase in sensitivity to apoptosis when compared to control ALX1165L / 165L NCC ( black ) . The data on the left represents the mean percentage of Annexin V positive cells , indicative of apoptosis , as determined by FACS analysis , with the data on the right being an example of one such experiment . Apoptosis was induced by immersion in a 55 ° C water bath for ten minutes . Representative experiment for each condition is shown . Data is represented as pooled mean ± SEM of three independent experiments . Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . . * : Significantly different from the basal apoptosis rate : p = 3x10 - 12 between control NCC basal apoptosis and induced apoptosis , and between ALX1165F / 165F NCC basal apoptosis and induced apoptosis . * * : Significantly different from control NCC ( p = 0 . 0004 ) . B . Expression levels of cyclins CCNA2 ( blue ) and CCND1 ( orange ) in NCC at passages 2 and 3 of ALX1165L / 165L and ALX1165F / 165F NCC . The RT - qPCR relative expression values were normalized to RPLP0 and GAPDH expression . Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype . * : Significantly different from control NCC at passage 2 ( p = 0 . 001 between control and ALX1165F / 165F NCC at passage 2 for CCNA2 , p = 0 . 0052 for CCND1 ) . * * : Significantly different from control NCC at passage 3 ( p = 0 . 0494 between control and ALX1165F / 165F NCC for CCNA2 , p = 0 . 0008 for CCND1 ) . C . Fluorescence Activated Cell Sorting ( FACS ) experiments showed that control ALX1165L / 165L NCC ( green ) exhibited increased expression of mesenchymal markers CD90 , CD105 , and CD73 with culture time ( passages 1 through 4 ) , whereas homozygous ALX1165F / 165F NCC ( blue ) showed a consistent expression of the markers expressed at passage 1 throughout . Further , control ALX1165L / 165L NCC showed a downregulation of CD57 expression with culture time , while ALX1165F / 165F NCC maintained the same level of CD57 across passages . Data are presented as the mean percentage of positive - stained cells across passage numbers . Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC . For CD90 , p = 0 . 0013 at passage 3 and p = 0 . 0207 at passage 4 . For CD105 , p = 0 . 0016 at passage 2 , p = 0 . 00004 at passage 3 and p = 0 . 0021 at passage 4 . For CD73 , p = 0 . 0060 at passage 2 , p = 0 . 00004 at passage 3 and p = 0 . 0114 at passage 4 . For CD57 , p = 0 . 0026 at passage 2 , p = 0 . 000003 at passage 3 and p = 0 . 000007 at passage 4 .

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

Figure 4A. Homozygous ALX1165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1165L/165L NCC (black). The data on the left represents the mean percentage of Annexin V positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for ten minutes. Representative experiment for each condition is shown. Data is represented as pooled mean ± SEM of three independent experiments. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. . *: Significantly different from the basal apoptosis rate: p=3x10-12 between control NCC basal apoptosis and induced apoptosis, and between ALX1165F/165F NCC basal apoptosis and induced apoptosis. **: Significantly different from control NCC (p=0.0004). B. Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1165L/165L and ALX1165F/165F NCC. The RT-qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data is represented as pooled mean ± SEM of three experiments on three clones from each genotype. *: Significantly different from control NCC at passage 2 (p=0.001 between control and ALX1165F/165F NCC at passage 2 for CCNA2, p=0.0052 for CCND1). **: Significantly different from control NCC at passage 3 (p=0.0494 between control and ALX1165F/165F NCC for CCNA2, p=0.0008 for CCND1).C. Fluorescence Activated Cell Sorting (FACS) experiments showed that control ALX1165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive-stained cells across passage numbers. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. *: Significantly different from control NCC. For CD90, p=0.0013 at passage 3 and p=0.0207 at passage 4. For CD105, p=0.0016 at passage 2, p=0.00004 at passage 3 and p=0.0021 at passage 4. For CD73, p=0.0060 at passage 2, p=0.00004 at passage 3 and p=0.0114 at passage 4. For CD57, p=0.0026 at passage 2, p=0.000003 at passage 3 and p=0.000007 at passage 4.


Figure 5A . Mutant ALX1165F / 165F NCC ( blue ) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L / 165L NCC ( black ) . Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24 - hour period . For fluorescent pictures , cells were stained in serum free media containing 3 . 6µM CellTracker Green CMFDA ( Life Technologies ) for 30 min at 37 ° C and allowed to recover for 30 min before starting the experiment . Images were acquired every 6 hours using a Keyence BZ - X800 microscope . Surface area analyses and percentages of coverage were measured using ImageJ software ( NIH ) . The NCC were monitored over 24 hours . The data are represented as the average of the percentage of closure ± SEM . Scale bar = 200µm . Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . * : Significantly different from control NCC ( p < 0 . 0001 ) . B . Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F / 165F NCC ( blue ) secrete less BMP2 and more BMP9 compared to control ALX1165L / 165L NCC ( green ) . Data are represented as pooled mean ± SEM of three clones from each genotype . Statistical significance was determined with an ANOVA test . A p - value < 0 . 05 was considered significant . * : Significantly different from control NCC ( p = 0 . 0424 for BMP2 and p = 0 . 0192 for BMP9 ) . C . Addition of soluble BMP2 or CV2 , a BMP9 antagonist , to the culture medium could partially rescue the migration defect of ALX1165F / 165F NCC . At the beginning of the assay , 10 , 50 , or 100 ng / ml of soluble BMP2 , CV2 , or a combination of the two at 100 ng / ml each were added to the culture medium , and the cells were monitored over the next 24 hours . The data are represented as the average of the percentage of closure ± SEM . Scale bar : 400µm .

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

Figure 5A. Mutant ALX1165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1165L/165L NCC (black). Data is presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24-hour period. For fluorescent pictures, cells were stained in serum free media containing 3.6µM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 hours using a Keyence BZ-X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200µm. Data obtained of each clone from three independent experiments were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant.*: Significantly different from control NCC (p<0.0001).B. Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1165F/165F NCC (blue) secrete less BMP2 and more BMP9 compared to control ALX1165L/165L NCC (green). Data are represented as pooled mean ± SEM of three clones from each genotype. Statistical significance was determined with an ANOVA test. A p-value <0.05 was considered significant. *: Significantly different from control NCC (p =0.0424 for BMP2 and p=0.0192 for BMP9).C. Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 hours. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400µm.


Figure 6A . Dissected flatmount wild type and alx1 - / - zebrafish larvae craniofacial cartilages after Alcian blue staining , the anterior points to the left of the page in all images . The ventral cartilages appear normal , but the alx1 - / - anterior neurocranium ( ANC ) appears narrow , with the midline element that is derived from the frontonasal NCC being absent . The Meckel ' s cartilage ( arrow , MC ) is also diminutive . Scale bar : 200 µm . B . Zebrafish alx1 mutants ( blue ) show reduced detectable expression of alx1 but increased expression of alx3 , alx4a compared to wild type controls ( green ) . alx4b expression levels are similar between wild type and alx1 - / - lines . Data is represented as the mean of all pooled embryos from three different clutches . The RT - qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method . Data from each clutch were pooled and the mathematical mean was calculated . SEM was used to determine the standard error . To test statistical significance , an ANOVA test was performed . A p - value < 0 . 05 was considered to be statistically significant . Statistical significance denoted by * ; p < 0 . 0001 between WT zebrafish and alx1 - / - at all measured time points ; at 10 ss , 24 hpf and 36 hpf for alx3 ; and at 24 hpf and 36 hpf for alx4a . Refer to Table EV2 for p - values . C . Dissected flatmount of zebrafish embryos injected with Alx1DN , after Alcian blue staining . The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium ( ANC ) and a profound hypoplasia of the Meckel ' s and ventral cartilages . In the most severely affected zebrafish , a nearly abrogated ANC was observed . Scale bar : 200 µm . D . Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted . In the control animal , the anterior cranial NCC always migrate to contribute to the median portion of the ANC . In contrast , the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC , where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location ( white asterisks ) . Scale bar : 250µm .

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

Figure 6A. Dissected flatmount wild type and alx1-/- zebrafish larvae craniofacial cartilages after Alcian blue staining, the anterior points to the left of the page in all images. The ventral cartilages appear normal, but the alx1-/- anterior neurocranium (ANC) appears narrow, with the midline element that is derived from the frontonasal NCC being absent. The Meckel's cartilage (arrow, MC) is also diminutive. Scale bar: 200 µm.B. Zebrafish alx1 mutants (blue) show reduced detectable expression of alx1 but increased expression of alx3, alx4a compared to wild type controls (green). alx4b expression levels are similar between wild type and alx1-/- lines. Data is represented as the mean of all pooled embryos from three different clutches. The RT-qPCR relative expression values were normalized to elfa and 18S expression using the ΔΔCT method. Data from each clutch were pooled and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A p-value <0.05 was considered to be statistically significant. Statistical significance denoted by *; p<0.0001 between WT zebrafish and alx1-/- at all measured time points; at 10 ss, 24 hpf and 36 hpf for alx3; and at 24 hpf and 36 hpf for alx4a. Refer to Table EV2 for p-values.C. Dissected flatmount of zebrafish embryos injected with Alx1DN, after Alcian blue staining. The embryos developed an absence of the frontonasal derived median portion of the anterior neurocranium (ANC) and a profound hypoplasia of the Meckel's and ventral cartilages. In the most severely affected zebrafish, a nearly abrogated ANC was observed. Scale bar: 200 µm.D. Lineage tracing experiments in control and Alx1DN mutant embryos revealed aberrant migration of anterior cranial NCC when alx1 is disrupted. In the control animal, the anterior cranial NCC always migrate to contribute to the median portion of the ANC. In contrast, the anterior cranial NCC labeled in the Alx1DN animals fail to migrate to the median ANC, where the ANC structure is narrower and the labeled cranial NCC are found in an anterior and lateral ectopic location (white asterisks). Scale bar: 250µm.


Figure 1Single plane confocal fluorescent microscopy images of 5 - day - old animals harboring GFP - tagged endogenous HSP - 110 ( HSP - 110 : : GFP ) . Muscle cells are outlined . M : muscle , H : hypodermis . In animals that co - express hairpin constructs targeting hsp - 110 in muscle cells ( HSP - 110 ; HPI and HSP - 110 ; HPII ) , GFP fluorescence is strongly reduced in muscle cells in comparison to control animals , indicating HSP - 110 : : GFP depletion specifically in muscle cells . Scale bar : 10 µm . Experimental set - up : Age - synchronized 4 - day - old animals were subjected to heat stress for 3 h at 33 ° C ( + HS ) then returned to 20 ° C or left at 20 ° C ( - HS ) , respectively . Arrows indicate imaging time points . The - HS controls were imaged at the same time point as the + HS animals after 24h at 20 ° C . Maximum intensity projections of fluorescent microscopy z - stacks of animals expressing the indicated transgenes . HS - induced FLUCSM : : GFP foci are cleared after a recovery for 24h at 20 ° C in the WT background , but persist in the presence of the hsp - 110 hairpin construct . Scale bar : 20 µm . C Quantification of FLUCSM : : GFP foci disaggregation . % disaggregation is calculated as 100 - ratio of FLUCSM : : GFP foci area relative to total muscle area at the + HS 24h time point to the + HS 2h time point . Data are displayed as mean ± SEM ( in % ) ( N = 3 ) . Statistical analysis was done using one - way ANOVA with Dunnett ' s adjustment for multiple comparisons . * * p ≤ 0 . 01 . Data information : All strains harbor the sid - 1 ( pk3321 ) allele .

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

Figure 1Single plane confocal fluorescent microscopy images of 5-day-old animals harboring GFP-tagged endogenous HSP-110 (HSP-110::GFP). Muscle cells are outlined. M: muscle, H: hypodermis. In animals that co-express hairpin constructs targeting hsp-110 in muscle cells (HSP-110;HPI and HSP-110;HPII), GFP fluorescence is strongly reduced in muscle cells in comparison to control animals, indicating HSP-110::GFP depletion specifically in muscle cells. Scale bar: 10 µm.Experimental set-up: Age-synchronized 4-day-old animals were subjected to heat stress for 3 h at 33°C (+HS) then returned to 20°C or left at 20°C (-HS), respectively. Arrows indicate imaging time points. The -HS controls were imaged at the same time point as the +HS animals after 24h at 20°C. Maximum intensity projections of fluorescent microscopy z-stacks of animals expressing the indicated transgenes. HS-induced FLUCSM::GFP foci are cleared after a recovery for 24h at 20°C in the WT background, but persist in the presence of the hsp-110 hairpin construct. Scale bar: 20 µm. C Quantification of FLUCSM::GFP foci disaggregation. % disaggregation is calculated as 100 - ratio of FLUCSM::GFP foci area relative to total muscle area at the +HS 24h time point to the +HS 2h time point. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's adjustment for multiple comparisons. ** p ≤ 0.01. Data information: All strains harbor the sid-1(pk3321) allele.


Figure 2A Maximum intensity projections of fluorescent microscopy z - stacks of 5 - day - old nematodes expressing the indicated transgenes are shown . White dashed lines outline the borders of muscle cells . Scale bar : 10 µm . The co - expression of HPI and HPII reduces the amount of α - Syn : : YFP inclusions . B Quantification of α - Syn : : YFP foci in 3 - to 6 - day - old animals . Product of mean foci fluorescence and foci area relative to muscle area is displayed ( relFluoFoci ) . Data are shown as mean ± SEM ( in % ) ( N = 3 ) . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . * * = p ≤ 0 . 01 , * * * p ≤ 0 . 001 . C Motility Assay as a measure for transgene toxicity . Displayed is the mean number of body bends per 30 s ± SEM ( N = 3 ) . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . D Maximum intensity projections of fluorescent microscopy z - stacks of 6 - day - old animals expressing the indicated transgenes . Scale bar : 100 µm . The co - expression of HPI and HPII reduces the amount of Q35 : : YFP inclusions . E Quantification of Q35 : : YFP foci in WT and hsp - 110 hairpin background . Shown is the mean ± SEM ( N = 3 ) . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . * p ≤ 0 . 05 , * * p ≤ 0 . 01 . F Motility Assay as a measure for transgene toxicity . Displayed is the mean number of body bends per 30 s ± SEM ( N = 3 ) . Statistical analysis was done using one - way ANOVA with Dunnett ' s multiple comparisons test . * p ≤ 0 . 05 , * * p ≤ 0 . 01 . G Maximum intensity projections of fluorescent microscopy z - stacks of of 5 - day - old nematodes expressing the indicated transgenes . White dashed lines outline the borders of muscle cells . Scale bar : 10 µm . M : muscle , H : hypodermis . Signal outside of muscle cells reveals spreading of α - Syn . H Quantification of animals showing α - Syn transmission at indicated ages . Displayed is the mean ± SEM ( in % ) ( N = 4 ) . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . * * * p ≤ 0 . 001 .

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

Figure 2A Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes are shown. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The co-expression of HPI and HPII reduces the amount of α-Syn::YFP inclusions. B Quantification of α-Syn::YFP foci in 3- to 6-day-old animals. Product of mean foci fluorescence and foci area relative to muscle area is displayed (relFluoFoci). Data are shown as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** = p ≤ 0.01, *** p ≤ 0.001. C Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. ** p ≤ 0.01, *** p ≤ 0.001.D Maximum intensity projections of fluorescent microscopy z-stacks of 6-day-old animals expressing the indicated transgenes. Scale bar: 100 µm. The co-expression of HPI and HPII reduces the amount of Q35::YFP inclusions.E Quantification of Q35::YFP foci in WT and hsp-110 hairpin background. Shown is the mean ± SEM (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. * p ≤ 0.05, ** p ≤ 0.01.F Motility Assay as a measure for transgene toxicity. Displayed is the mean number of body bends per 30 s ± SEM (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01.G Maximum intensity projections of fluorescent microscopy z-stacks of of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. M: muscle, H: hypodermis. Signal outside of muscle cells reveals spreading of α-Syn. H Quantification of animals showing α-Syn transmission at indicated ages. Displayed is the mean ± SEM (in %) (N = 4). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001.


Figure 3A Maximum intensity projections of confocal microscopy z - stacks of 5 - day - old animals expressing the indicated transgenes . White dashed lines outline the borders of muscle cells . Scale bar : 10 µm . The expression of the yeast SSE1N575Y E575A mutant cannot reverse the reduction of α - Syn : : YFP foci in the hsp - 110 hairpin expressing animals . B Quantification of motility as a measure for transgene toxicity . Displayed is the mean ± SEM ( N = 3 ) amount of body bends / 30s of animals expressing the indicated transgenes . Statistical analysis was done using two - way ANOVA and with Dunnett ' s multiple comparison test . n . s . = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . The presence of the yeast SSE1N575Y E575A mutant was not able to suppress the rescue of α - Syn : : YFP toxicity in hsp - 110 hairpin expressing animals . C Maximum intensity projections of fluorescent microscopy z - stacks of 5 - day - old nematodes expressing the indicated transgenes . White dashed lines outline the borders of muscle cells . M : muscle , H : hypodermis . Scale bars : 10 µm . Signal outside of muscle cells reveals spreading of α - Syn . D Quantification of animals showing α - Syn transmission into the hypodermis at indicated ages . Displayed is the mean ± SEM ( in % ) ( N = 3 ) . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . n . s . = not significant , * * * p ≤ 0 . 001 . E Experimental set - up : Age - synchronized 4 - day - old animals were subjected to heat stress for 3 h at 33 ° C ( + HS ) then returned to 20 ° C or left at 20 ° C ( - HS ) , respectively . Arrows indicate imaging time points . The - HS controls were imaged at the same time point as the + HS animals after 24h at 20 ° C . Maximum intensity projections of fluorescent microscopy z - stacks of animals expressing the indicated transgenes . Scale bar : 20 µm . The impaired clearance of HS - induced FLUCSM : : GFP foci in the hsp - 110 hairpin expressing cells is rescued by co - expression of WT but not by the HSP - 70 binding deficient SSE1N575Y E575A mutant . F Quantification of FLUCSM : : GFP foci disaggregation . % disaggregation is calculated as 100 - ratio of FLUCSM : : GFP foci area relative to total muscle area at the + HS 24h time point to the + HS 2h time point . Data are displayed as mean ± SEM ( in % ) ( N = 3 ) . Statistical analysis was done using one - way ANOVA with Dunnett ' s multiple comparison test . n . s . = not significant , * * p ≤ 0 . 01 . All strains harbor the sid - 1 ( pk3321 ) allele .

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

Figure 3A Maximum intensity projections of confocal microscopy z-stacks of 5-day-old animals expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. Scale bar: 10 µm. The expression of the yeast SSE1N575Y E575A mutant cannot reverse the reduction of α-Syn::YFP foci in the hsp-110 hairpin expressing animals.B Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes. Statistical analysis was done using two-way ANOVA and with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001. The presence of the yeast SSE1N575Y E575A mutant was not able to suppress the rescue of α-Syn::YFP toxicity in hsp-110 hairpin expressing animals.C Maximum intensity projections of fluorescent microscopy z-stacks of 5-day-old nematodes expressing the indicated transgenes. White dashed lines outline the borders of muscle cells. M: muscle, H: hypodermis. Scale bars: 10 µm. Signal outside of muscle cells reveals spreading of α-Syn.D Quantification of animals showing α-Syn transmission into the hypodermis at indicated ages. Displayed is the mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001.E Experimental set-up: Age-synchronized 4-day-old animals were subjected to heat stress for 3 h at 33°C (+HS) then returned to 20°C or left at 20°C (-HS), respectively. Arrows indicate imaging time points. The -HS controls were imaged at the same time point as the +HS animals after 24h at 20°C. Maximum intensity projections of fluorescent microscopy z-stacks of animals expressing the indicated transgenes. Scale bar: 20 µm. The impaired clearance of HS-induced FLUCSM::GFP foci in the hsp-110 hairpin expressing cells is rescued by co-expression of WT but not by the HSP-70 binding deficient SSE1N575Y E575A mutant.F Quantification of FLUCSM::GFP foci disaggregation. % disaggregation is calculated as 100 - ratio of FLUCSM::GFP foci area relative to total muscle area at the +HS 24h time point to the +HS 2h time point. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01. All strains harbor the sid-1(pk3321) allele.


Figure 4A Maximum intensity projections of confocal z - stacks of 4 - day - old animals expressing the indicated transgenes or harboring the indicated temperature - sensitive ( ts ) mutations . Muscle cells were stained with anti - myosin ( green ) and Alexa Flour 647 - phalloidin ( purple ) . Scale bar : 5 µm . Myosin misfolding reflects the exposure of the ts mutant phenotype of myosin ( ts ) [ unc - 54 ( e1301 ) ] at the permissive temperate of 15 ° C by co - expression of the hsp - 110 hairpins . B Quantification of motility as a measure for toxicity . Displayed is the mean ± SEM ( N = 4 ) of body bends / 30s of animals expressing the indicated transgenes or harboring the indicated mutations . Statistical analysis was done using one - way ANOVA with Dunnett ' s multiple comparison test . * * * p ≤ 0 . 001 . Co - expression of the hsp - 110 hairpins exposed the ts mutant phenotype at the permissive temperate of 15 ° C , resulting in a significant increase in movement defects . All strains contain the sid - 1 ( pk3321 ) allele . C Quantification of motility as a measure for transgene toxicity . Displayed is the mean ± SEM ( N = 4 ) amount of body bends / 30s of animals expressing the indicated transgenes or harboring the indicated mutations . Statistical analysis was done using two - way ANOVA with Tukey ' s multiple comparison test . * p ≤ 0 . 05 . Expression of the hsp - 110 hairpins caused movement defects with increasing age . D Maximum intensity projections of confocal z - stacks of 12 - day - old animals expressing the indicated transgenes . Scale bar : 10 µm . FLUCSM forms foci with increasing age , which is markedly accelerated upon hsp - 110 KD . E Quantification of animals with FLUCSM : : GFP foci at indicated days of life . Data are displayed as mean ± SEM ( in % ) ( N = 3 ) . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . n . s . = not significant , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . F Quantification of motility as a measure for transgene toxicity . Displayed is the mean ± SEM ( N = 3 ) amount of body bends / 30s of animals expressing the indicated transgenes or harboring the indicated mutations . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . n . s . = not significant , * * * p ≤ 0 . 001 . Expression of the hsp - 110 hairpins caused movement defects with increasing age . The beneficial effects of HSP - 110 KD on amyloid toxicity is masked in older animals . G Quantification of motility as a measure for transgene toxicity . Displayed is the mean ± SEM ( N = 3 ) amount of body bends / 30s of animals expressing the indicated transgenes or harboring the indicated mutations . Statistical analysis was done using two - way ANOVA with Dunnett ' s multiple comparison test . n . s . = not significant , , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . Expression of the hsp - 110 hairpins caused movement defects with increasing age . The beneficial effects of HSP - 110 KD on amyloid toxicity is masked in older animals . Data information : All strains harbor the sid - 1 ( pk3321 ) allele .

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

Figure 4A Maximum intensity projections of confocal z-stacks of 4-day-old animals expressing the indicated transgenes or harboring the indicated temperature-sensitive (ts) mutations. Muscle cells were stained with anti-myosin (green) and Alexa Flour 647-phalloidin (purple). Scale bar: 5 µm. Myosin misfolding reflects the exposure of the ts mutant phenotype of myosin(ts) [unc-54(e1301)] at the permissive temperate of 15°C by co-expression of the hsp-110 hairpins.B Quantification of motility as a measure for toxicity. Displayed is the mean ± SEM (N = 4) of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using one-way ANOVA with Dunnett's multiple comparison test. *** p ≤ 0.001. Co-expression of the hsp-110 hairpins exposed the ts mutant phenotype at the permissive temperate of 15°C, resulting in a significant increase in movement defects. All strains contain the sid-1(pk3321) allele.C Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 4) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Tukey's multiple comparison test. * p ≤ 0.05. Expression of the hsp-110 hairpins caused movement defects with increasing age.D Maximum intensity projections of confocal z-stacks of 12-day-old animals expressing the indicated transgenes. Scale bar: 10 µm. FLUCSM forms foci with increasing age, which is markedly accelerated upon hsp-110 KD.E Quantification of animals with FLUCSM::GFP foci at indicated days of life. Data are displayed as mean ± SEM (in %) (N = 3). Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, ** p ≤ 0.01, *** p ≤ 0.001.F Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals.G Quantification of motility as a measure for transgene toxicity. Displayed is the mean ± SEM (N = 3) amount of body bends/30s of animals expressing the indicated transgenes or harboring the indicated mutations. Statistical analysis was done using two-way ANOVA with Dunnett's multiple comparison test. n.s. = not significant, , ** p ≤ 0.01, *** p ≤ 0.001. Expression of the hsp-110 hairpins caused movement defects with increasing age. The beneficial effects of HSP-110 KD on amyloid toxicity is masked in older animals. Data information: All strains harbor the sid-1(pk3321) allele.


f2 ( b ) The CD spectra of the Beclin 1 CC domain at different temperatures . ( c ) . Dynamic exchange assay of wild - type Beclin 1 . His - tagged and untagged Beclin 1 CC domain were co - incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads . The presence of heterodimeric Beclin 1 CC domain , consisting of one monomer with his - tag and the other monomer without tag , was checked by SDS gel and indicated by the arrow . ( d ) Self - association pattern of Beclin 1 in vivo . Both GFP - and AsRed - tagged full - length Beclin 1 are co - transfected into HEK 293T cells and their self - association is probed by co - IP using GFP antibody for immunoprecipitation , followed by western blot with anti - AsRed antibody .

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

f2(b) The CD spectra of the Beclin 1 CC domain at different temperatures.(c). Dynamic exchange assay of wild-type Beclin 1. His-tagged and untagged Beclin 1 CC domain were co-incubated at the indicated temperatures and coprecipitated at preset temperatures by Ni2+- nitrilotriacetic acid (NTA) agarose beads. The presence of heterodimeric Beclin 1 CC domain, consisting of one monomer with his-tag and the other monomer without tag, was checked by SDS gel and indicated by the arrow.(d) Self-association pattern of Beclin 1 in vivo. Both GFP- and AsRed-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for immunoprecipitation, followed by western blot with anti-AsRed antibody.


f3 ( a , b ) ITC titration profile of Beclin 1 CC domain with Atg14L ( a ) or UVRAG ( b ) . c ) Pull - down assay to assess the competition between Beclin 1 - Atg14L and Beclin 1 - UVRAG complexes . Increasing amount of His6 - tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region . The Beclin 1 - Atg14L / UVRAG complexes were pulled down by Ni2 + - nitrilotriacetic acid ( NTA ) agarose beads and checked by SDS gel . ( d ) Co - IP experiments to assess the competition between Beclin 1 - Atg14L and Beclin 1 - UVRAG complexes in vivo . FLAG - or GFP - tagged Atg14L or UVRAG was transfected alone , or both were co - transfected into HEK 293T cells and their interaction with endogenous Beclin 1 was probed by co - IP using GFP antibody , followed by western blot with anti - Beclin 1 antibody .

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f3(a, b) ITC titration profile of Beclin 1 CC domain with Atg14L (a) or UVRAG (b).c) Pull-down assay to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes. Increasing amount of His6-tagged Beclin 1 CC domain was added to a mixture of Atg14L and UVRAG proteins corresponding to their respective CC region. The Beclin 1-Atg14L/UVRAG complexes were pulled down by Ni2+- nitrilotriacetic acid (NTA) agarose beads and checked by SDS gel.(d) Co-IP experiments to assess the competition between Beclin 1-Atg14L and Beclin 1-UVRAG complexes in vivo. FLAG- or GFP-tagged Atg14L or UVRAG was transfected alone, or both were co-transfected into HEK 293T cells and their interaction with endogenous Beclin 1 was probed by co-IP using GFP antibody, followed by western blot with anti-Beclin 1 antibody.


f4 ( c ) Self - association pattern of wild - type , MutM and MutStab constructs of Beclin 1 in vivo . For each construct , both GFP - and Myc - tagged full - length Beclin 1 are co - transfected into HEK 293T cells and their self - association is probed by co - IP using GFP antibody for IP , followed by western blot with anti - Myc antibody . ( d ) . Temperature - dependent CD profile of Beclin 1 CC domain wild - type and MutStab constructs at 220 nm .

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f4(c) Self-association pattern of wild-type, MutM and MutStab constructs of Beclin 1 in vivo. For each construct, both GFP- and Myc-tagged full-length Beclin 1 are co-transfected into HEK 293T cells and their self-association is probed by co-IP using GFP antibody for IP, followed by western blot with anti-Myc antibody.(d). Temperature-dependent CD profile of Beclin 1 CC domain wild-type and MutStab constructs at 220 nm.


f5 ( a , b ) Interaction of Beclin 1 wild - type and monomeric ( MutM ) mutants with Atg14L or UVRAG in cultured cells . HEK 293T cells were transfected with GFP - tagged Beclin 1 variant alone ( a ) or together with FLAG - UVRAG ( b ) . The lysates were immunoprecipitated by GFP antibody , followed by western - blot analysis with anti - Atg14L antibody for detecting binding to endogenous Atg14L ( a ) or with anti - UVRAG antibody for detecting binding to the FLAG - tagged UVRAG protein ( b ) .

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f5(a,b) Interaction of Beclin 1 wild-type and monomeric (MutM) mutants with Atg14L or UVRAG in cultured cells. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (a) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western-blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).


f6 ( a , b ) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo . HEK 293T cells were transfected with GFP - tagged Beclin 1 variant alone ( b ) or together with FLAG - UVRAG ( b ) . The lysates were immunoprecipitated by GFP antibody , followed by western blot analysis with anti - Atg14L antibody for detecting binding to endogenous Atg14L ( a ) or with anti - UVRAG antibody for detecting binding to the FLAG - tagged UVRAG protein ( b ) .

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f6(a, b) Interaction of MutStab mutants of Beclin 1 with Atg14L or UVRAG in vivo. HEK 293T cells were transfected with GFP-tagged Beclin 1 variant alone (b) or together with FLAG-UVRAG (b). The lysates were immunoprecipitated by GFP antibody, followed by western blot analysis with anti-Atg14L antibody for detecting binding to endogenous Atg14L (a) or with anti-UVRAG antibody for detecting binding to the FLAG-tagged UVRAG protein (b).


f7 ( a , b ) . Representative fluorescent images of HeLa cells co - tansfected with GFP - tagged Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( green ) and AsRed - Atg14L ( red ) ( a ) or FLAG - UVRAG ( red ) ( b ) ; Co - localization of AsRed - Atg14L with GFP - tagged Beclin 1 wild type or MutM is shown in discrete puncta . ( c - e ) Representative fluorescence images of HeLa cells co - transfected with Myc - Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( blue ) , AsRed - Atg14L ( red ) and GFP - tagged Atg12 ( c ) . The scale bar is 20 μM . ( c - e ) Representative fluorescence images of HeLa cells co - transfected with Myc - Beclin 1 wild type , MutM L178A / L192A or MutStab E189L / A217L / E224L / A255L ( blue ) , AsRed - Atg14L ( red ) and GFP - tagged LC3 ( d ) and DFCP ( e ) ( green ) . The scale bar is 20 μM .

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

f7(a,b). Representative fluorescent images of HeLa cells co-tansfected with GFP-tagged Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (green) and AsRed-Atg14L (red) (a) or FLAG-UVRAG (red) (b); Co-localization of AsRed-Atg14L with GFP-tagged Beclin 1 wild type or MutM is shown in discrete puncta.(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged Atg12 (c). The scale bar is 20 μM.(c-e) Representative fluorescence images of HeLa cells co-transfected with Myc-Beclin 1 wild type, MutM L178A/L192A or MutStab E189L/A217L/E224L/A255L (blue), AsRed-Atg14L (red) and GFP-tagged LC3 (d) and DFCP (e) (green). The scale bar is 20 μM.


Figure 1 ( A ) Hematoxylin and Eosin ( H & E ) staining of WT and K14ΔNLef1 HF from back skin at 11 . 5 weeks ( early anagen ) . White arrowheads : ectopic SG and epidermal cysts . Data information Scale bars : 50 μm . ( B ) Tail epidermal whole - mounts from WT and K14ΔNLef1 mice labelled with Fabp5 , Krt15 and counterstained with Dapi . Data information : Scale bars : 50 μm . ( C ) Average signal intensity of ΔNLef1 binding sites detected by a Lef1 antibody ( red ) as compared to input alone ( grey ) and negative control ( Neg . Ctl . , black ) . DNA motif analysis revealed Lef1 classical consensus sequence . Representative plots of ChIP - Seq reads aligned to the Vgll4 , Runx1 and Gata6 loci . ( D ) Heat - map ( Pearson correlation ) of differentially expressed genes ( DEG ) between different micro - dissected regions ( IFE , HF and SG ) from WT mice ( left panel ) ΔNLef1 Chip - seq data show ΔNLef1 direct transcriptional targets in black ( right panel ) . ( E ) Gene Set Enrichment Analysis comparing gene location of ΔNLef1 peaks and SG gene expression signature . ( F ) RT - qPCR analysis of Gata6 in WT and K14ΔNLef1 primary keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled sequence ( scr . ) . Data are means ± SEM of three independent wells . Data information Statistical analyses were performed with an ordinary one - way ANOVA : ( ns ) not significant ; ( * ) p - value < 0 . 05 ; ( * * * ) p - value < 0 . 0005 . ( G ) RT - qPCR analysis of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1 - or ΔN34Lef1 - expressing plasmid . ∆ N34Lef1 is the human ortholog of murine ∆ N32Lef1 , which is expressed in K14 ∆ NLef1 transgenic mice Data are means ± SEM of three independent experiments . Data information : Statistical analyses were performed with an ordinary one - way ANOVA : ( ns ) not significant ; ( * ) p - value < 0 . 05 ; ( * * * ) p - value < 0 . 0005 . ( H ) Dorsal skin sections of WT and K14ΔNLef1 mice stained for Krt14 and Gata6 . Data information Scale bars : 50 μm . ( I ) Heat - maps of DEG from WT - or K14ΔNLef1 - unfractionated ( all ) or sorted ( Itga6 + Cd34 - , basal ; Itga6 + Cd34 + , HFSC ) keratinocytes in comparison with IFE , SG , and HF gene signatures ranked from high to low expression . ( J ) Heat - map depicting similarities , as Pearson ' s correlation coefficient , between DEG from keratinocytes from WT or K14ΔNLef1 mice and IFE vs SG vs HF transcriptome analysis ( left panel ) . Additional correlation with ΔNLef1 Chip - seq data is depicted in the right heat - map .

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

Figure 1(A) Hematoxylin and Eosin (H&E) staining of WT and K14ΔNLef1 HF from back skin at 11.5 weeks (early anagen). White arrowheads: ectopic SG and epidermal cysts. Data information Scale bars: 50 μm.(B) Tail epidermal whole-mounts from WT and K14ΔNLef1 mice labelled with Fabp5, Krt15 and counterstained with Dapi. Data information: Scale bars: 50 μm.(C) Average signal intensity of ΔNLef1 binding sites detected by a Lef1 antibody (red) as compared to input alone (grey) and negative control (Neg. Ctl., black). DNA motif analysis revealed Lef1 classical consensus sequence. Representative plots of ChIP-Seq reads aligned to the Vgll4, Runx1 and Gata6 loci.(D) Heat-map (Pearson correlation) of differentially expressed genes (DEG) between different micro-dissected regions (IFE, HF and SG) from WT mice (left panel) ΔNLef1 Chip-seq data show ΔNLef1 direct transcriptional targets in black (right panel).(E) Gene Set Enrichment Analysis comparing gene location of ΔNLef1 peaks and SG gene expression signature.(F) RT-qPCR analysis of Gata6 in WT and K14ΔNLef1 primary keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled sequence (scr.). Data are means ± SEM of three independent wells. Data information Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.(G) RT-qPCR analysis of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1- or ΔN34Lef1-expressing plasmid. ∆N34Lef1 is the human ortholog of murine ∆N32Lef1, which is expressed in K14∆NLef1 transgenic mice Data are means ± SEM of three independent experiments. Data information: Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.(H) Dorsal skin sections of WT and K14ΔNLef1 mice stained for Krt14 and Gata6. Data information Scale bars: 50 μm.(I) Heat-maps of DEG from WT- or K14ΔNLef1-unfractionated (all) or sorted (Itga6+Cd34-, basal; Itga6+Cd34+, HFSC) keratinocytes in comparison with IFE, SG, and HF gene signatures ranked from high to low expression.(J) Heat-map depicting similarities, as Pearson's correlation coefficient, between DEG from keratinocytes from WT or K14ΔNLef1 mice and IFE vs SG vs HF transcriptome analysis (left panel). Additional correlation with ΔNLef1 Chip-seq data is depicted in the right heat-map.


Figure 2 ( A ) Sections of WT embryonic skin at different HF stages stained for Gata6 , Lef1 , Sox9 and counterstained with Dapi . Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis . Quantification of the percentage of cells labelled for both Gata6 and Lef1 , or Gata6 and Sox9 in stages 4 - 5 HF are shown ( upper right panel ) . Data are means ± SD and were obtained from 9 HF from 3 mice . ( B ) Sections of E18 . 5 WT and Lef1 - / - mouse skin stained for Lef1 and Gata6 . Deletion of Lef1 does not impair Gata6 expression . Data information : Scale bars : 50 μm . ( C ) Sections of WT and K14ΔNβcateninER ( K14ΔNβ - catER D2 and D4 strains ) adult dorsal skin stained with antibodies against Krt14 and Gata6 . Topical treatment with 1 or 6 doses of 4OHT activates β - catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression . Data information : Scale bars : 50 μm . ( D ) Sections of E18 . 5 WT and K14Cre / βCat Flox ( ex3 ) / + mouse skin stained for Sox9 and Gata6 . Activation of Wnt / β - catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of the HFSC marker Sox9 . Data information : Scale bar : 25 μm .

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Figure 2(A) Sections of WT embryonic skin at different HF stages stained for Gata6, Lef1, Sox9 and counterstained with Dapi. Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis. Quantification of the percentage of cells labelled for both Gata6 and Lef1, or Gata6 and Sox9 in stages 4-5 HF are shown (upper right panel). Data are means ± SD and were obtained from 9 HF from 3 mice. (B) Sections of E18.5 WT and Lef1-/- mouse skin stained for Lef1 and Gata6. Deletion of Lef1 does not impair Gata6 expression. Data information: Scale bars: 50 μm.(C) Sections of WT and K14ΔNβcateninER (K14ΔNβ-catER D2 and D4 strains) adult dorsal skin stained with antibodies against Krt14 and Gata6. Topical treatment with 1 or 6 doses of 4OHT activates β-catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression. Data information: Scale bars: 50 μm.(D) Sections of E18.5 WT and K14Cre/βCat Flox(ex3)/+ mouse skin stained for Sox9 and Gata6. Activation of Wnt/β-catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of the HFSC marker Sox9. Data information: Scale bar: 25 μm.


Figure 3 ( A ) Bright - field images of whole - mount WT and Gata6 cKO epidermis , showing abnormal SG upon loss of Gata6 . Quantification of hypotrophic SG in WT , heterozygous ( Het ) and Gata6 cKO epidermal whole - mounts . Data are boxplots with indication of means . Box limits are minimum and maximum values . An average of 165 HF per mouse ( from 3 to 4 mice per genotype ) was analyzed . ( * ) p - value < 0 . 05 ; ( * * ) p - value < 0 . 005 ; unpaired Student ' s t - test . Data information : Scale bars : 50 μm . ( B ) Schematic representation of plasmid constructs used for in utero lentiviral infection . Whole - mounts or sections of adult infected epidermis with empty vector ( EV ) or Gata6 - ires - GFP ( G6OE ) lentivirus were stained for GFP and Fasn . In vivo overexpression of Gata6 leads to ectopic Fasn expression in the HF / SG unit . White dotted lines define SG , and yellow dotted lines define a cyst . Note that the cyst is mostly negative for Fasn in agreement with its SD - like phenotype . White arrows indicate GFP - positive infected cells . These cells are stained with Fasn only in G6OE condition . H & E - stained skin from G6OE mice shows a cyst with SG elements in the HF unit ( black arrows ) . Staining for Gata6 ( both endogenous and exogenous ) shows that Gata6 expression occurs in a limited number of cells ( representative image in upper right panel ) . Bottom left graph shows quantification of the percentage of clones labelled for both Fasn and GFP in the SG , HF and IFE compartments . Data are means ± SD and originate from 4 EV mice and 8 G6OE mice ( average of 11 clones per mouse ) . ( C ) Whole - mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX . Ectopic Gata6 cells are not stained with LipidTOX indicating an incomplete sebaceous maturation . Data information : Scale bars : 50 μm . ( D ) Lineage tracing experiments in Gata6EGFPCreERT2 : Rosa26 - fl / STOP / fl - tdTomato ( Gata6creER ROSA - dTom ) mice . A single dose of 4OHT was injected in pregnant females at E16 . 5 . Tail skin from pups was collected at P13 . Representative example of whole - mount epidermis showing tdTomato labelled cells counterstained with Dapi ( top left panel ) . Right panels show the different Z - stacks related to this whole - mount . Gata6 progenies are mainly found in the upper SG / JZ . Localization of Gata6 progenies ( dTomato + ) was quantified in 20 - 26 pilosebaceous units per mouse ( N = 4 mice ) ( bottom left panel ) . Data are means ± SD . As a control , quantification was also performed in Rosa26 - fl / STOP / fl - tdTomato ( ROSA - dTom ) mice that were treated similarly to Gata6creER ROSA - dTomato mice . Data information : Scale bar : 25 μm .

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

Figure 3(A) Bright-field images of whole-mount WT and Gata6 cKO epidermis, showing abnormal SG upon loss of Gata6. Quantification of hypotrophic SG in WT, heterozygous (Het) and Gata6 cKO epidermal whole-mounts. Data are boxplots with indication of means. Box limits are minimum and maximum values. An average of 165 HF per mouse (from 3 to 4 mice per genotype) was analyzed. (*) p-value < 0.05; (**) p-value < 0.005; unpaired Student's t-test. Data information: Scale bars: 50 μm.(B) Schematic representation of plasmid constructs used for in utero lentiviral infection. Whole-mounts or sections of adult infected epidermis with empty vector (EV) or Gata6-ires-GFP (G6OE) lentivirus were stained for GFP and Fasn. In vivo overexpression of Gata6 leads to ectopic Fasn expression in the HF/SG unit. White dotted lines define SG, and yellow dotted lines define a cyst. Note that the cyst is mostly negative for Fasn in agreement with its SD-like phenotype. White arrows indicate GFP-positive infected cells. These cells are stained with Fasn only in G6OE condition. H&E-stained skin from G6OE mice shows a cyst with SG elements in the HF unit (black arrows). Staining for Gata6 (both endogenous and exogenous) shows that Gata6 expression occurs in a limited number of cells (representative image in upper right panel). Bottom left graph shows quantification of the percentage of clones labelled for both Fasn and GFP in the SG, HF and IFE compartments. Data are means ± SD and originate from 4 EV mice and 8 G6OE mice (average of 11 clones per mouse). (C) Whole-mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX. Ectopic Gata6 cells are not stained with LipidTOX indicating an incomplete sebaceous maturation. Data information: Scale bars: 50 μm.(D) Lineage tracing experiments in Gata6EGFPCreERT2:Rosa26-fl/STOP/fl-tdTomato (Gata6creER ROSA-dTom) mice. A single dose of 4OHT was injected in pregnant females at E16.5. Tail skin from pups was collected at P13. Representative example of whole-mount epidermis showing tdTomato labelled cells counterstained with Dapi (top left panel). Right panels show the different Z-stacks related to this whole-mount. Gata6 progenies are mainly found in the upper SG/JZ. Localization of Gata6 progenies (dTomato+) was quantified in 20-26 pilosebaceous units per mouse (N=4 mice) (bottom left panel). Data are means ± SD. As a control, quantification was also performed in Rosa26-fl/STOP/fl-tdTomato (ROSA-dTom) mice that were treated similarly to Gata6creER ROSA-dTomato mice. Data information: Scale bar: 25 μm.