( A ) Indicated strains were spotted 5 - fold dilutions on synthetic complete ( SC ) plates , and plates were incubated at room temperature , 30oC , and 37oC ( 3 biological replicates , 2 technical replicates ; n = 5 ) . WT , dfm1 ∆ , tsc3 ∆ , and dfm1 ∆ tsc3 ∆ were compared for growth in the dilution assay . Arrowhead indicates growth phenotype of tsc3 ∆ cells ; open circle indicates growth phenotype of dfm1 ∆ tsc3 ∆ cells . ( B ) dfm1 ∆ tsc3 ∆ confers resistance to myrocin . WT , dfm1 ∆ , tsc3 ∆ , and dfm1 ∆ tsc3 ∆ strains were grown to log - phase in YPD medium , and 5 - fold serial dilutions of cultures were spotted on ( SC ) plates containing either drug vehicle alone , 1 µM of myriocin and 10 μM of PHS ( 3 biological replicates , 2 technical replicates ; n = 5 ) . Arrowhead indicates growth phenotype of tsc3 ∆ cells ; open circle indicates growth phenotype of dfm1 ∆ tsc3 ∆ cells .

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

(A) Indicated strains were spotted 5-fold dilutions on synthetic complete (SC) plates , and plates were incubated at room temperature, 30oC, and 37oC (3 biological replicates, 2 technical replicates; n=5). WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ were compared for growth in the dilution assay. Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells. (B) dfm1∆tsc3∆ confers resistance to myrocin. WT, dfm1∆, tsc3∆, and dfm1∆tsc3∆ strains were grown to log-phase in YPD medium, and 5-fold serial dilutions of cultures were spotted on (SC) plates containing either drug vehicle alone, 1 µM of myriocin and 10 μM of PHS (3 biological replicates, 2 technical replicates; n=5). Arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of dfm1∆tsc3∆ cells.


( D ) Indicated strains were spotted 5 - fold dilutions on SC plates in 3 biological replicates and 2 technical replicates ( n = 5 ) , and plates were incubated at room temperature , 30oC , and 37oC ( n = 3 ) . Upper panel : WT , orm1 ∆ , tsc3 ∆ , and orm1 ∆ tsc3 ∆ were compared for growth by dilution assay . Middle panel : WT , orm2 ∆ , tsc3 ∆ , and orm2 ∆ tsc3 ∆ were compared for growth by dilution assay . Bottom panel : WT , orm1 ∆ , orm2 ∆ , tsc3 ∆ , and orm1 ∆ orm2 ∆ tsc3 ∆ , were compared for growth by dilution assay . Upper panel : arrowhead indicates growth phenotype of tsc3 ∆ cells ; open circle indicates growth phenotype of orm1 ∆ tsc3 ∆ cells . Lower panel : arrowhead indicates growth phenotype of orm2 ∆ cells ; open circle indicates growth phenotype of orm2 ∆ tsc3 ∆ cells .

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

(D) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC (n=3). Upper panel: WT, orm1∆, tsc3∆, and orm1∆tsc3∆ were compared for growth by dilution assay. Middle panel: WT, orm2∆, tsc3∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, orm1∆, orm2∆, tsc3∆, and orm1∆orm2∆tsc3∆, were compared for growth by dilution assay. Upper panel: arrowhead indicates growth phenotype of tsc3∆ cells; open circle indicates growth phenotype of orm1∆tsc3∆ cells. Lower panel: arrowhead indicates growth phenotype of orm2∆ cells; open circle indicates growth phenotype of orm2∆tsc3∆ cells.


( A ) Indicated strains were spotted 5 - fold dilutions on SC plates in 3 biological replicates and 2 technical replicates ( n = 5 ) , and plates were incubated at room temperature , 30oC , and 37oC . Upper panel : WT , dfm1 ∆ , orm1 ∆ , and dfm1 ∆ orm1 ∆ were compared for growth by dilution assay . Middle panel : WT , dfm1 ∆ , orm2 ∆ , and orm2 ∆ tsc3 ∆ were compared for growth by dilution assay . Bottom panel : WT , dfm1 ∆ , orm1 ∆ , orm2 ∆ , and orm2 ∆ , and orm1 ∆ orm2 ∆ were compared for growth by dilution assay . Upper , middle , and lower panel : gray arrowhead depicts growth phenotype of dfm1 ∆ orm1 ∆ , dfm1 ∆ orm2 ∆ , and orm1 ∆ orm2 ∆ respectively . ( B ) dfm1 ∆ orm1 ∆ confers resistance to myriocin and sensitivity to PHS . WT , dfm1 ∆ , orm1 ∆ , and dfm1 ∆ orm1 ∆ strains were grown to log - phase in SC medium , and 5 - fold serial dilutions of cultures were spotted on YPD plates containing either drug vehicle alone , 1 mM of myriocin and 10 μM of PHS with each condition performed in 3 biological replicates and 2 technical replicates ( n = 5 ) . Plates were incubated at room temperature and photographed after 3 days . Open circle indicates growth phenotype of dfm1 ∆ orm1 ∆ cells .

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

(A) Indicated strains were spotted 5-fold dilutions on SC plates in 3 biological replicates and 2 technical replicates (n=5), and plates were incubated at room temperature, 30oC, and 37oC. Upper panel: WT, dfm1∆, orm1∆, and dfm1∆orm1∆ were compared for growth by dilution assay. Middle panel: WT, dfm1∆, orm2∆, and orm2∆tsc3∆ were compared for growth by dilution assay. Bottom panel: WT, dfm1∆, orm1∆, orm2∆, and orm2∆, and orm1∆orm2∆ were compared for growth by dilution assay. Upper, middle, and lower panel: gray arrowhead depicts growth phenotype of dfm1∆orm1∆, dfm1∆orm2∆, and orm1∆orm2∆ respectively. (B) dfm1∆orm1∆ confers resistance to myriocin and sensitivity to PHS. WT, dfm1∆, orm1∆, and dfm1∆orm1∆ strains were grown to log-phase in SC medium, and 5-fold serial dilutions of cultures were spotted on YPD plates containing either drug vehicle alone, 1 mM of myriocin and 10 μM of PHS with each condition performed in 3 biological replicates and 2 technical replicates (n=5). Plates were incubated at room temperature and photographed after 3 days. Open circle indicates growth phenotype of dfm1∆orm1∆ cells.


( A ) Degradation of Orm2 depends on Dfm1 and not Der1 . The indicated strains expressing Orm2 - RFP were grown into log phase and degradation was measured by cycloheximide chase ( CHX ) . After CHX addition , cells were lysed at the indicated times , and analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ( n = 3 ) . ( B ) Same as ( A ) except degradation of Orm2 - RFP was measured in WT and der1 ∆ cells ( 3 biological replicates ; n = 3 ) .

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

(A) Degradation of Orm2 depends on Dfm1 and not Der1. The indicated strains expressing Orm2-RFP were grown into log phase and degradation was measured by cycloheximide chase (CHX). After CHX addition, cells were lysed at the indicated times, and analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates (n=3). (B) Same as (A) except degradation of Orm2-RFP was measured in WT and der1∆ cells (3 biological replicates; n=3).


( D ) Dfm1 ' s SHP box is not required for degradation of Orm2 - RFP . In the indicated strains , degradation of Orm2 - RFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ; n = 3 ) .

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

(D) Dfm1's SHP box is not required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).


( G ) Dfm1 ' s WR motif , GxxxG motif , substrate binding and lipid thinning function are required for degradation of Orm2 - RFP . In the indicated strains , degradation of Orm2 - RFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ; n = 3 ) .

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

(G) Dfm1's WR motif, GxxxG motif, substrate binding and lipid thinning function are required for degradation of Orm2-RFP. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3).


( H ) Dfm1 L1 residues are required for binding to Orm2 . Orm2 - RFP and binding to retrotranslocation - deficient Dfm1 L1 mutants was analyzed by co - IP . The IP was analyzed for presence of Dfm1 - HA . As a negative control , cells not expressing Orm2 - RFP were used ( 3 biological replicates ; n = 3 ) . Band intensities for all western blots were normalized to PGK1 loading control and quantified by ImageJ .

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

(H) Dfm1 L1 residues are required for binding to Orm2. Orm2-RFP and binding to retrotranslocation-deficient Dfm1 L1 mutants was analyzed by co-IP. The IP was analyzed for presence of Dfm1-HA. As a negative control, cells not expressing Orm2-RFP were used (3 biological replicates; n=3). Band intensities for all western blots were normalized to PGK1 loading control and quantified by ImageJ.


( A ) E3 ligase Tul1 is required for Orm2 degradation . In the indicated strains , degradation of Orm2 - RFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted for Orm2 - RFP with α - RFP ( 3 biological replicates ; n = 3 ) . t = 0 was taken as 100 % and data is represented as mean ± SEM .

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

(A) E3 ligase Tul1 is required for Orm2 degradation. In the indicated strains, degradation of Orm2-RFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted for Orm2-RFP with α-RFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.


( B ) Cdc48 and the proteasome are required for Orm2 degradation . Same as ( A ) , except cdc48 - 2 and hrd2 - 1 were analyzed for Orm2 - RFP degradation ( 3 biological replicates ; n = 3 ) . t = 0 was taken as 100 % and data is represented as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cdc48 and the proteasome are required for Orm2 degradation. Same as (A), except cdc48-2 and hrd2-1 were analyzed for Orm2-RFP degradation (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.


( C ) Dfm1 does not function in the post - ubiquitination step of Orm2 degradation pathway . Indicated strains expressing Orm2 - RFP were grown into log phase . Cells were lysed , and microsomes were collected and immunoprecipitated with α - RFP conjugated to agarose beads . Samples were then subjected to SDS - PAGE and immunoblot by α - Ubiquitin and α - RFP ( 3 biological replicates ; n = 3 ) .

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

(C) Dfm1 does not function in the post-ubiquitination step of Orm2 degradation pathway. Indicated strains expressing Orm2-RFP were grown into log phase. Cells were lysed, and microsomes were collected and immunoprecipitated with α-RFP conjugated to agarose beads. Samples were then subjected to SDS-PAGE and immunoblot by α-Ubiquitin and α-RFP (3 biological replicates; n=3).


( D ) ERAD mutants do not rescue temperature - sensitive lethality of tsc3 ∆ . Indicated strains were grown to log - phase in SC and serially diluted cultures were plated on SC plates and incubated at 37 ° C and imaged at Day 2 ( 3 biological replicates , 2 technical replicates ; n = 5 ) .

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

(D) ERAD mutants do not rescue temperature-sensitive lethality of tsc3∆. Indicated strains were grown to log-phase in SC and serially diluted cultures were plated on SC plates and incubated at 37°C and imaged at Day 2 (3 biological replicates, 2 technical replicates; n=5).


( A ) Phosphorylated Orm2 accumulates in the absence of Dfm1 . Phos - tag western blot analysis shows that there is an accumulation of phosphorylated Orm2 in dfm1 ∆ cells . The indicated strains were grown to log phase , treated with vehicle or 1 . 5 μM myriocin for 1 hour and subjected to SDS - PAGE or Phos - tag western blot analysis via blotting for Orm2 with α - RFP and PGK1 with α - PGK1 antibodies ( 2 biological replicates ; n = 2 ) .

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

(A) Phosphorylated Orm2 accumulates in the absence of Dfm1. Phos-tag western blot analysis shows that there is an accumulation of phosphorylated Orm2 in dfm1∆ cells. The indicated strains were grown to log phase, treated with vehicle or 1.5 μM myriocin for 1 hour and subjected to SDS-PAGE or Phos-tag western blot analysis via blotting for Orm2 with α-RFP and PGK1 with α-PGK1 antibodies (2 biological replicates; n=2).


( C ) dfm1 ∆ cells block the degradation of phosphorylated mimic of Orm2 ( Orm2 - 3D ) . In the indicated strains , degradation of Orm2 - 3A - GFP and Orm2 - 3D - GFP was measured by CHX - chase assay . Cells were analyzed by SDS - PAGE and immunoblotted α - GFP ( 3 biological replicates ; n = 3 ) . t = 0 was taken as 100 % and data is represented as mean ± SEM .

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

(C) dfm1∆ cells block the degradation of phosphorylated mimic of Orm2 (Orm2-3D). In the indicated strains, degradation of Orm2-3A-GFP and Orm2-3D-GFP was measured by CHX-chase assay. Cells were analyzed by SDS-PAGE and immunoblotted α-GFP (3 biological replicates; n=3). t=0 was taken as 100% and data is represented as mean ± SEM.


( A ) Dfm1 - HA binding to Orm2 - GFP , Orm2 - 3A - GFP , Orm2 - 3D - GFP , Orm1 , Lcb1 , and Lcb2 were analyzed by co - IP . As negative control , cells not expressing Dfm1 - HA were used . Also , Sec61 was also included to test for non - specific binding ( 3 biological replicates ; n = 3 ) . ( B ) Same as ( A ) , except co - IP was performed on WT and orm2 ∆ cells and Dfm1 - HA binding to Orm1 , Lcb1 , and Lcb2 was analyzed ( 3 biological replicates ; n = 3 ) . ( C ) Same as ( A ) , except Orm2 - GFP binding to Dfm1 - HA , Orm1 , Lcb1 , and Lcb2 was analyzed by co - IP ( 3 biological replicates ; n = 3 ) .

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

(A) Dfm1-HA binding to Orm2-GFP, Orm2-3A-GFP, Orm2-3D-GFP, Orm1, Lcb1, and Lcb2 were analyzed by co-IP. As negative control, cells not expressing Dfm1-HA were used. Also, Sec61 was also included to test for non-specific binding (3 biological replicates; n=3). (B) Same as (A), except co-IP was performed on WT and orm2∆ cells and Dfm1-HA binding to Orm1, Lcb1, and Lcb2 was analyzed (3 biological replicates; n=3). (C) Same as (A), except Orm2-GFP binding to Dfm1-HA, Orm1, Lcb1, and Lcb2 was analyzed by co-IP (3 biological replicates; n=3).


( A ) Cellulase functional screening by Congo Red assay . Halos on the plate indicate cellulase activity . Pc : Pseudomonas cellulosa , Bs : Bacillus subtilis . Numbered strains are from the C . elegans native gut

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

(A) Cellulase functional screening by Congo Red assay. Halos on the plate indicate cellulase activity. Pc: Pseudomonas cellulosa, Bs: Bacillus subtilis. Numbered strains are from the C. elegans native gut microbiome


( B ) Production of reducing sugars by cellulose - degrading bacterial strains . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Error bars represent 95 % confidence intervals of the mean .

O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Production of reducing sugars by cellulose-degrading bacterial strains. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.


( D ) Colony - forming units ( CFU ) of various bacterial species in the worm gut . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Error bars represent 95 % confidence intervals of the mean .

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

(D) Colony-forming units (CFU) of various bacterial species in the worm gut. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.


( E ) C . elegans colonized with cellulolytic P . cellulosa and incubated with different carbon sources . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Error bars represent 95 % confidence intervals of the mean .

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

(E) C. elegans colonized with cellulolytic P. cellulosa and incubated with different carbon sources. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Error bars represent 95% confidence intervals of the mean.


( A ) Incorporation of 14C - labelled cellulose . Worms were first colonized with cellulolytic bacteria and then incubated with 14C - cellulose to allow carbon utilization . Antibiotic treatment was applied before 14C measurement of worms to eliminate bacterial interference in isotopic reading . 14C - cellulose incorporation measurement reflected the isotopic carbon incorporation in C . elegans . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Student ' s t test was used for statistical analysis . In A , experiments were performed in biological triplicates using 200 worms per data point . In A error bars represent 95 % confidence intervals for the mean .

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

(A) Incorporation of 14C-labelled cellulose. Worms were first colonized with cellulolytic bacteria and then incubated with 14C-cellulose to allow carbon utilization. Antibiotic treatment was applied before 14C measurement of worms to eliminate bacterial interference in isotopic reading. 14C-cellulose incorporation measurement reflected the isotopic carbon incorporation in C. elegans. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In A, experiments were performed in biological triplicates using 200 worms per data point. In A error bars represent 95% confidence intervals for the mean.


( B ) Induction of the worm fat - 7p : : GFP metabolic reporter . Adult worms pre - colonized with P . cellulosa were incubated in liquid media for 24 h with the indicated carbon sources , and GFP fluorescence was read in individual worms on a BioSorter large object sorter . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Student ' s t test was used for statistical analysis . In B , error bars represent 95 % confidence intervals for the mean . In B , each point represents a different worm in biological replicates , n = 96 , n = 53 , and n = 80 from left to right .

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

(B) Induction of the worm fat-7p::GFP metabolic reporter. Adult worms pre-colonized with P. cellulosa were incubated in liquid media for 24 h with the indicated carbon sources, and GFP fluorescence was read in individual worms on a BioSorter large object sorter. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In B, error bars represent 95% confidence intervals for the mean. In B, each point represents a different worm in biological replicates, n=96, n=53, and n=80 from left to right.


( C ) Effect of nutrition on larval yield . Pre - colonized worms were separated into the individual wells of a 384 - well plate , and larvae counts in each well were read after 48 h . Larval yield comparison indicates the nutritional benefit of cellulolytic activity . Data information : All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis . Student ' s t test was used for statistical analysis . In C , the central band represents the median , notches represent 95 % confidence intervals for the median , the box edges represent the inter quartile range ( IQR ) and the whiskers extend an additional 1 . 5 IQR beyond the boxes . In C , each point represents a different worm in biological replicates , n = 74 , n = 79 , n = 79 , n = 89 , n = 88 , n = 86 from left to right .

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

(C) Effect of nutrition on larval yield. Pre-colonized worms were separated into the individual wells of a 384-well plate, and larvae counts in each well were read after 48 h. Larval yield comparison indicates the nutritional benefit of cellulolytic activity. Data information: All experiments were performed in biological triplicates. Student's t test was used for statistical analysis. Student's t test was used for statistical analysis. In C, the central band represents the median, notches represent 95% confidence intervals for the median, the box edges represent the inter quartile range (IQR) and the whiskers extend an additional 1.5 IQR beyond the boxes. In C, each point represents a different worm in biological replicates, n=74, n=79, n=79, n=89, n=88, n=86 from left to right.


Synchronized adult worms were colonized for two days on plates containing E . coli OP50 + P . cellulosa ( Pc ) , E . coli OP50 + Lp , or E . coli OP50 + Pc + Lp . ( B ) After 36 h outgrowth of the pathogen , worms pre - colonized with P . cellulosa in the presence of L . plantarum ( E . coli OP50 + Pc + Lp ) showed lower pathogen burden than P . cellulosa monocolonized worms when CMC was used as the carbon source . Data shown are fold change Salmonella infection ( CFU / worm ) relative to the P . cellulosa - only condition . Error bars represent 95 % confidence interval of the mean . CMC = 0 . 01 % carboxymethylcellulose ( w / v ) . All experiments were performed in biological triplicates . Student ' s t test was used for statistical analysis .

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

Synchronized adult worms were colonized for two days on plates containing E. coli OP50 + P. cellulosa (Pc), E. coli OP50 + Lp, or E. coli OP50 + Pc + Lp. (B) After 36 h outgrowth of the pathogen, worms pre-colonized with P. cellulosa in the presence of L. plantarum (E. coli OP50 + Pc + Lp) showed lower pathogen burden than P. cellulosa monocolonized worms when CMC was used as the carbon source. Data shown are fold change Salmonella infection (CFU/worm) relative to the P. cellulosa-only condition. Error bars represent 95% confidence interval of the mean. CMC = 0.01% carboxymethylcellulose (w/v). All experiments were performed in biological triplicates. Student's t test was used for statistical analysis.


B . Total quantified proteome depth in tissues ( n = 18 ) , primary cells ( n = 12 ) , immortalized cell lines ( n = 4 ) and all samples ( n = 34 ) . In all cases , n means biological replicates unless otherwise indicated . The upper - and lower panel shows the number of quantified protein groups and peptides , respectively . C . Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank ( Liver : bulk liver biopsy , HepA : hepatic artery , PorV : portal vein , Cell lines : mixture of human liver - derived immortalized cell lines ) . Number of biological replicates is same as Panel ( A ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Total quantified proteome depth in tissues (n=18), primary cells (n=12), immortalized cell lines (n=4) and all samples (n=34). In all cases, n means biological replicates unless otherwise indicated. The upper- and lower panel shows the number of quantified protein groups and peptides, respectively. C. Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank (Liver: bulk liver biopsy, HepA: hepatic artery, PorV: portal vein, Cell lines: mixture of human liver-derived immortalized cell lines). Number of biological replicates is same as Panel (A).


B . Cumulative protein abundance of liver biopsy , haptic artery and portal vein as a function of protein rank , with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated .

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

B. Cumulative protein abundance of liver biopsy, haptic artery and portal vein as a function of protein rank, with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated.


D . Cumulative protein abundance of liver cell types as a function of protein rank , indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles .

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

D. Cumulative protein abundance of liver cell types as a function of protein rank, indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles.


F . The bar plot shows the contribution of each organelle to total cellular protein mass , also accounting for unassigned proteins . Percentage of cytosol , mitochondria , nucleoli , plasma membrane and actin filaments is indicated .

F. The bar plot shows the contribution of each organelle to total cellular protein mass, also accounting for unassigned proteins. Percentage of cytosol, mitochondria, nucleoli, plasma membrane and actin filaments is indicated.


A . Total protein copy number of the oxidative phosphorylation complex I - V in bulk liver tissue ( n = 6 independent biological replicates ) and primary cell types ( isolated primary cells from n = 3 individuals for each cell type ) . Values are presented as mean ± s . d . B . Protein copy number for members of the oxidative phosphorylation complex I - V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated .

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

A. Total protein copy number of the oxidative phosphorylation complex I-V in bulk liver tissue (n=6 independent biological replicates) and primary cell types (isolated primary cells from n=3 individuals for each cell type). Values are presented as mean ± s.d. B. Protein copy number for members of the oxidative phosphorylation complex I-V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated.


D . Top ten uniquely quantified proteins per cell type with LFQ intensities [ Log10 ] and copy numbers [ Log10 ] .

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

D. Top ten uniquely quantified proteins per cell type with LFQ intensities [Log10] and copy numbers [Log10].


A . Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types , with columns showing three biological replicates ( n = 3 ) of four cell types and rows significant proteins . Significance was calculated by one - way ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Frames and numbers indicate four clusters of proteins highly enriched in each cell type .

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

A. Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types, with columns showing three biological replicates (n=3) of four cell types and rows significant proteins. Significance was calculated by one-way ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Frames and numbers indicate four clusters of proteins highly enriched in each cell type.


A . Total quantified proteins at day one , three and seven upon primary cell culture in hepatocytes ( hHEP ) , hepatic stellate cells ( hHSC ) and liver sinusoidal endothelial cells ( LSEC ) . In all cases , number of biological replicates is 3 . Values are presented as mean ± s . d . No additional replications of the experiment was done in laboratory .

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

A. Total quantified proteins at day one, three and seven upon primary cell culture in hepatocytes (hHEP), hepatic stellate cells (hHSC) and liver sinusoidal endothelial cells (LSEC). In all cases, number of biological replicates is 3. Values are presented as mean ± s.d. No additional replications of the experiment was done in laboratory.


F . Protein expression patterns over the course of primary hHSC culture ( n = 3 ) . Values are presented as mean ± s . d . Significance was calculated by ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * P < 0 . 01 , and * * * P < 0 . 001 .

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

F. Protein expression patterns over the course of primary hHSC culture (n=3). Values are presented as mean ± s.d. Significance was calculated by ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of **P < 0.01, and ***P < 0.001.


A . Hierarchical clustering of proteins significantly differentially abundant between NASH ( n = 20 ) , cirrhosis ( n = 10 ) and control groups ( n = 15 ) . Significance was calculated by ANCOVA , followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated . No additional replications of the experiment was done in laboratory .

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

A. Hierarchical clustering of proteins significantly differentially abundant between NASH (n=20), cirrhosis (n=10) and control groups (n=15). Significance was calculated by ANCOVA, followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated. No additional replications of the experiment was done in laboratory.


D . Box ‐ and ‐ whisker plot showing the distribution of log2 ‐ intensity values of statistical significantly regulated proteins across three groups . Number of independent biological replicates is n = 15 , 20 and 10 for the control , NASH and cirrhosis group , respectively . The black line in the middle of the box is the median , the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers ( Q3 + 1 . 5 * IQR , Q1 ‐ 1 . 5 * IQR ) where IQR is the interquartile range ( Q3 - Q1 ) . Significance was defined by ANCOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * * P < 0 . 001 . E . Heatmap of CYP 450 family members that are statistically significantly abundant between three groups . Data was presented as mean protein abundance followed by Z - score normalization across . Number of replicates is same as Panel ( C ) . F . Extracellular matrix ( ECM ) remodeling in liver cirrhosis . Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control ( upper ) and cirrhosis ( lower ) groups , highlighting top shifted ECM components in the cirrhosis group as compared with the control group .

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

D. Box‐and‐whisker plot showing the distribution of log2‐intensity values of statistical significantly regulated proteins across three groups. Number of independent biological replicates is n=15, 20 and 10 for the control, NASH and cirrhosis group, respectively. The black line in the middle of the box is the median, the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers (Q3+1.5*IQR, Q1‐1.5*IQR) where IQR is the interquartile range (Q3-Q1). Significance was defined by ANCOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of ***P < 0.001. E. Heatmap of CYP 450 family members that are statistically significantly abundant between three groups. Data was presented as mean protein abundance followed by Z-score normalization across. Number of replicates is same as Panel (C). F. Extracellular matrix (ECM) remodeling in liver cirrhosis. Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control (upper) and cirrhosis (lower) groups, highlighting top shifted ECM components in the cirrhosis group as compared with the control group.


A . Lysates of HK2 cells cultured in FM or STV ( 2h ) were immunoprecipitated with anti - OFD1 ( left ) and anti - ATG13 ( right ) antibodies or IgG and analyzed by WB ; ACTIN was used as loading control . n = 3 independent experiments . IP = immunoprecipitation , FM = full medium , STV = HBSS starvation .

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

A. Lysates of HK2 cells cultured in FM or STV(2h) were immunoprecipitated with anti-OFD1 (left) and anti-ATG13 (right) antibodies or IgG and analyzed by WB; ACTIN was used as loading control. n=3 independent experiments. IP=immunoprecipitation, FM=full medium, STV=HBSS starvation.


B . Airyscan confocal analysis of endogenous FIP200 / γTUBULIN and ATG13 / γTUBULIN colocalization with lentiviral delivered HA - OFD1 in HK2 cells . Cells were cultured in HBSS ( 90min ) . Green , FIP200 and ATG13 ; red , HA - OFD1 ; Grey , γTUBULIN ; blue , Hoechst for nuclei . Insets show magnifications and single - color channels of selected areas . Scale bar = 3 , 5μm . C . Quantification of HA - OFD1 / ATG13 and HA - OFD1 / FIP200 colocalization , expressed as % of total OFD1 fluorescence ( mean ± SEM ) . n = 3 independent experiments ; n = 53 cells for ATG13 and n = 39 for FIP200 were analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Airyscan confocal analysis of endogenous FIP200/γTUBULIN and ATG13/γTUBULIN colocalization with lentiviral delivered HA-OFD1 in HK2 cells. Cells were cultured in HBSS (90min). Green, FIP200 and ATG13; red, HA-OFD1; Grey, γTUBULIN; blue, Hoechst for nuclei. Insets show magnifications and single-color channels of selected areas. Scale bar=3,5μm. C. Quantification of HA-OFD1/ATG13 and HA-OFD1/FIP200 colocalization, expressed as % of total OFD1 fluorescence (mean ± SEM). n=3 independent experiments; n=53 cells for ATG13 and n=39 for FIP200 were analyzed.


A . Top , WB of ULK1 - complex components in wt and KO - OFD1 cells assessed in FM ( - ) or STV ( 2 , 4h ) . Bands at same exposure , noncontiguous on the same gel . Bottom , ACTIN - normalized protein levels are expressed as fold increase compared with wt cells , represented by the dashed line ( mean ± SEM ) ; n = 5 independent experiments . FM = full medium , STV = HBSS starvation .

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

A. Top, WB of ULK1-complex components in wt and KO-OFD1 cells assessed in FM (-) or STV (2,4h). Bands at same exposure, noncontiguous on the same gel. Bottom, ACTIN-normalized protein levels are expressed as fold increase compared with wt cells, represented by the dashed line (mean ± SEM); n=5 independent experiments. FM=full medium, STV=HBSS starvation.


B . Top , wild - type and KO - OFD1 cells were incubated with 50µg / ml CHX . Immunoblots were probed with indicated antibodies . Bottom , quantification of βTUBULIN and ACTIN - normalized protein levels versus untreated conditions ( - ) , data are expressed as mean ± SEM ; n = 4 independent experiments . CHX = Cycloheximide , ns = not significative .

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

B. Top, wild-type and KO-OFD1 cells were incubated with 50µg/ml CHX. Immunoblots were probed with indicated antibodies. Bottom, quantification of βTUBULIN and ACTIN-normalized protein levels versus untreated conditions (-), data are expressed as mean ± SEM; n=4 independent experiments. CHX=Cycloheximide, ns=not significative.


C . Immunofluorescence of ULK1 and ATG13 puncta in wt and KO - OFD1 cells cultured in HBSS ( 90min ) . Green , ATG13 and ULK1 ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) Graphs display quantification of puncta / cell , data are expressed as mean ± SEM ; n = 5 independent experiments , n ≥ 200 cells / antibody .

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

C. Immunofluorescence of ULK1 and ATG13 puncta in wt and KO-OFD1 cells cultured in HBSS (90min). Green, ATG13 and ULK1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) Graphs display quantification of puncta/cell, data are expressed as mean ± SEM; n=5 independent experiments, n≥200 cells/antibody.


D . Western Blot of ULK1 complex components in KO - OFD1 cells transfected with empty vector ( Empty ) or 3xFLAG - OFD1 ( OFD1 ) . Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM . n = 5 independent experiments .

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

D. Western Blot of ULK1 complex components in KO-OFD1 cells transfected with empty vector (Empty) or 3xFLAG-OFD1 (OFD1). Histograms show quantification of band intensities normalized versus ACTIN expressed as mean ± SEM. n=5 independent experiments.


E . Western Blot of ULK1 - complex components in KO - OFD1 cells transfected with 3xFLAG - OFD1 ( OFD1 ) or empty vector ( Empty ) and treated ( + ) or not ( - ) with SAR405 ( 10μM , 6h ) . ACTIN is the loading control . On the right , ATG13 levels are expressed as fold increase compared with empty vector ( mean ± SEM ) ; n = 3 independent experiments .

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

E. Western Blot of ULK1-complex components in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or empty vector (Empty) and treated (+) or not (-) with SAR405 (10μM, 6h). ACTIN is the loading control. On the right, ATG13 levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.


F . Representative confocal images of co - staining of 3xFLAG - OFD1 with ATG13 in KO - OFD1 cells incubated in HBSS , treated ( + ) or not ( - ) with Baf - A1 ( 100nM , 90min ) . Green , ATG13 ; red , 3xFLAG - OFD1 ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) ATG13 puncta / cell are quantified and expressed as mean ± SEM ; n = 3 independent experiments , n ≥ 100 cells .

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

F. Representative confocal images of co-staining of 3xFLAG-OFD1 with ATG13 in KO-OFD1 cells incubated in HBSS, treated (+) or not (-) with Baf-A1(100nM, 90min). Green, ATG13; red, 3xFLAG-OFD1; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=3 independent experiments, n≥100 cells.


A . Middle . Lysates from 3xFLAG - OFD1 overexpressing HEK293 cells were added to beads with immobilized GST - fused LC3B , LC3BF52A - V53AdG , dNLC3BdG and GST alone . Right . Lysates from 3xFLAG - OFD1 ( OFD1 ) and empty vector ( Empty ) overexpressing HEK293 cells were added to GST - GABARAP - L1 and GST . Left . WB of total lysates ( input ) . All panels display WB for FLAG , ACTIN is the loading control .

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

A. Middle. Lysates from 3xFLAG-OFD1 overexpressing HEK293 cells were added to beads with immobilized GST-fused LC3B, LC3BF52A-V53AdG, dNLC3BdG and GST alone. Right. Lysates from 3xFLAG-OFD1(OFD1) and empty vector (Empty) overexpressing HEK293 cells were added to GST-GABARAP-L1 and GST. Left. WB of total lysates (input). All panels display WB for FLAG, ACTIN is the loading control.


B . Representative confocal images of endogenous OFD1 ( green ) co - staining with LC3B ( red ) , and of 3xFLAG - OFD1 ( red ) with GABARAP ( green ) in HK2 cells ( HBSS , 90min ) . Hoechst labels nuclei ( blue ) . Insets show magnifications and single - color channels of selected areas . Scale bar = 10μm ; n ≥ 50 cells / sample .

B. Representative confocal images of endogenous OFD1 (green) co-staining with LC3B (red), and of 3xFLAG-OFD1 (red) with GABARAP (green) in HK2 cells (HBSS,90min). Hoechst labels nuclei (blue). Insets show magnifications and single-color channels of selected areas. Scale bar=10μm; n≥50 cells/sample.


D . Lysates from 3xFLAG - OFD1 ( OFD1 ) , 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) and empty vector ( Empty ) overexpressing cells were added to GST - GABARAP - L1 , GST - LC3B and GST .

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

D. Lysates from 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1∆LIR(OFD1∆LIR) and empty vector (Empty) overexpressing cells were added to GST-GABARAP-L1, GST-LC3B and GST.


E . Representative confocal images of OFD1 ∆ LIR co - staining with LC3B ( left ) and GABARAP ( right ) in HK2 cells ( HBSS , 90min ) . Green , LC3B and GABARAP ; red , 3xFLAG - OFD1 ∆ LIR ; blue , Hoechst for nuclei . Scale bar = 5μm . n ≥ 50 cells / sample .

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

E. Representative confocal images of OFD1∆LIR co-staining with LC3B (left) and GABARAP (right) in HK2 cells (HBSS,90min). Green, LC3B and GABARAP; red, 3xFLAG-OFD1∆LIR; blue, Hoechst for nuclei. Scale bar=5μm. n≥50 cells/sample.


A . Representative confocal images of 3xFLAG - OFD1 ( OFD1 ) and 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) co - staining with ATG13 in KO - OFD1 cells ( HBSS , 90min ) . Green , ATG13 ; red , FLAG ; blue , Hoechst for nuclei . Scale bar = 10μm . ( Right ) ATG13 puncta / cell are quantified and expressed as mean ± SEM ; n = 4 independent experiments , n ≥ 100 cells .

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

A. Representative confocal images of 3xFLAG-OFD1 (OFD1) and 3xFLAG-OFD1∆LIR (OFD1∆LIR) co-staining with ATG13 in KO-OFD1 cells (HBSS,90min). Green, ATG13; red, FLAG; blue, Hoechst for nuclei. Scale bar=10μm. (Right) ATG13 puncta/cell are quantified and expressed as mean ± SEM; n=4 independent experiments, n≥100 cells.


B . Western blot of ATG13 in KO - OFD1 cells transfected with 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) or empty vector ( Empty ) . ( Right ) ACTIN - normalized ATG13 protein levels are expressed as fold increase compared with empty vector ( mean ± SEM ) ; n = 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot of ATG13 in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) or empty vector (Empty). (Right) ACTIN-normalized ATG13 protein levels are expressed as fold increase compared with empty vector (mean ± SEM); n=3 independent experiments.


C . Top . OFD1 fragments ( a , b and c ) . Bottom left . Lysates from empty vector ( Empty ) , 3xFLAG - OFD1 ( OFD1 ) , 3xFLAG - OFD1a ( OFD1a ) , 3xFLAG - OFD1b ( OFD1b ) , 3xFLAG - OFD1c ( OFD1c ) overexpressing HEK293 cells were added to GST - ATG13 ; lysates from 3xFLAG - OFD1 ( OFD1 ) overexpressing HEK293 cells were added to GST as control . Right . WB of total lysates ( input ) . All panels display WB for FLAG , HSP90 is the loading control . LIR = LC3 interacting region .

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

C. Top. OFD1 fragments (a,b and c). Bottom left. Lysates from empty vector (Empty), 3xFLAG-OFD1(OFD1), 3xFLAG-OFD1a (OFD1a), 3xFLAG-OFD1b (OFD1b), 3xFLAG-OFD1c (OFD1c) overexpressing HEK293 cells were added to GST-ATG13; lysates from 3xFLAG-OFD1(OFD1) overexpressing HEK293 cells were added to GST as control. Right. WB of total lysates (input). All panels display WB for FLAG, HSP90 is the loading control. LIR=LC3 interacting region.


D . Scanning confocal microscopy analysis of wt and KO - OFD1 cells incubated in HBSS ( 8h ) , treated ( + ) or not ( - ) with Baf - A1 ( 50nM , 8h ) . Green , LAMP1 ; red , ATG13 ; blue , Hoechst for nuclei . Scale bar = 6μm . Insets show higher magnification and single colour channels of the boxed area . ( Bottom ) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1 / cell ( mean ± SEM ) . n = 30 WT cells , n = 28 KO - OFD1 cells counted ; three independent experiments .

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D. Scanning confocal microscopy analysis of wt and KO-OFD1 cells incubated in HBSS (8h), treated (+) or not (-) with Baf-A1(50nM, 8h). Green, LAMP1; red, ATG13; blue, Hoechst for nuclei. Scale bar=6μm. Insets show higher magnification and single colour channels of the boxed area. (Bottom) Bar graphs show quantification of lysosomes containing ATG13 expressed as % of total amount of LAMP1/cell (mean ± SEM). n=30 WT cells, n=28 KO-OFD1 cells counted; three independent experiments.


E . Time course of OFD1 levels in HBSS - starved wt HK2 cells treated with Baf - A1 ( 100nM ) and collected at different time points . LC3B is the control for Baf - A1 treatment . ( Right ) Graphs show ACTIN - normalized OFD1 levels versus FM condition ( - ) expressed as mean ± SEM ; n = 4 independent experiments . STV = starvation , Baf - A1 = bafilomycin , Ctrl = control .

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E. Time course of OFD1 levels in HBSS-starved wt HK2 cells treated with Baf-A1 (100nM) and collected at different time points. LC3B is the control for Baf-A1 treatment. (Right) Graphs show ACTIN-normalized OFD1 levels versus FM condition (-) expressed as mean ± SEM; n=4 independent experiments. STV=starvation, Baf-A1=bafilomycin, Ctrl=control.


F . Western blot of FLAG in KO - OFD1 cells transfected with 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) and treated or not ( - ) with Baf - A1 ( 100nM , 2h ) . ( Right ) Quantification of GAPDH - normalized FLAG levels versus untreated ( - ) is expressed as mean ± SEM ; n = 3 independent experiments . Baf - A1 = bafilomycin .

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F. Western blot of FLAG in KO-OFD1 cells transfected with 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) and treated or not (-) with Baf-A1 (100nM,2h). (Right) Quantification of GAPDH-normalized FLAG levels versus untreated (-) is expressed as mean ± SEM; n=3 independent experiments. Baf-A1=bafilomycin.


A . Representative blots of ATG13 ( left ) and VPS34 ( right ) phosphorylation levels in wt and KO - OFD1 cells transiently expressing GFP - ATG13 and FLAG - VPS34 in FM .

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A. Representative blots of ATG13 (left) and VPS34 (right) phosphorylation levels in wt and KO-OFD1 cells transiently expressing GFP-ATG13 and FLAG-VPS34 in FM.


B . Wild - type and KO - OFD1 cells were starved in HBSS with / without Baf - A1 ( 100nM , 2h ) and chloroquine ( CQ ) ( 50μM , 2h ) and total lysates were analyzed by immunoblotting using anti - OFD1 , - LC3B ( LC3B - I 18 kDa and LC3B - II 16 kDa ) and - ACTIN antibodies . ( Right ) ACTIN - normalized LC3B - II levels are expressed as fold change versus untreated conditions ( - ) of wt cells . n = 5 independent experiments .

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B. Wild-type and KO-OFD1 cells were starved in HBSS with/without Baf-A1 (100nM, 2h) and chloroquine (CQ) (50μM, 2h) and total lysates were analyzed by immunoblotting using anti-OFD1, -LC3B (LC3B-I 18 kDa and LC3B-II 16 kDa) and -ACTIN antibodies. (Right) ACTIN-normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of wt cells. n=5 independent experiments.


C . Representative images of LC3B and WIPI2 puncta in wt and KO - OFD1 cells ( HBSS , 90min ) . Green , LC3B ; red , WIPI2 ; blue , Hoechst , nuclei . Scale bar = 10μm . ( Right ) Quantification of LC3B and WIPI2 puncta is shown . ≥ 100 cells analyzed / sample , n = 5 independent experiments .

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C. Representative images of LC3B and WIPI2 puncta in wt and KO-OFD1 cells (HBSS, 90min). Green, LC3B; red, WIPI2; blue, Hoechst, nuclei. Scale bar=10μm. (Right) Quantification of LC3B and WIPI2 puncta is shown. ≥100 cells analyzed/sample, n=5 independent experiments.


D . Transmission electron microscopy ( left ) and quantification ( right ) of autophagic vacuoles ( arrows ) in wt and KO - OFD1 cells ( HBSS , 90min ) .

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D. Transmission electron microscopy (left) and quantification (right) of autophagic vacuoles (arrows) in wt and KO-OFD1 cells (HBSS, 90min).


E . Representative images of siOFD1 depleted and control ( Ctrl ) HeLa cells transiently expressing mRFP - eGFP - LC3B ( HBSS , 90min ) . Insets show higher magnification of colocalization in selected areas . Scale bar = 10μm . Bottom , tandem structure of the mRFP - GFP - LC3B construct and quantification of RFP + GFP + and RFP + GFP - puncta in siOFD1 - depleted cells and controls , cultured in full medium ( FM ) or kept in HBSS for 2h ( STV ) are depicted . n ≥ 90 cells / sample analyzed , n = 5 independent experiments .

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E. Representative images of siOFD1 depleted and control (Ctrl) HeLa cells transiently expressing mRFP-eGFP-LC3B (HBSS, 90min). Insets show higher magnification of colocalization in selected areas. Scale bar=10μm. Bottom, tandem structure of the mRFP-GFP-LC3B construct and quantification of RFP+GFP+ and RFP+GFP- puncta in siOFD1-depleted cells and controls, cultured in full medium (FM) or kept in HBSS for 2h (STV) are depicted. n≥90 cells/sample analyzed, n=5 independent experiments.


F . Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP - OFD1 ( HBSS , 90min ) . Green , eGFP - OFD1 , red , WIPI2 ; blue , Hoechst labels nuclei . On the right , quantification of WIPI2 puncta in eGFP - OFD1 transfected cells compared with non - transfected cells ( Ctrl ) is shown . Scale bar = 10μm . n ≥ 90 cells analyzed / sample , n = 3 independent experiments .

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F. Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP-OFD1 (HBSS,90min). Green, eGFP-OFD1, red, WIPI2; blue, Hoechst labels nuclei. On the right, quantification of WIPI2 puncta in eGFP-OFD1 transfected cells compared with non-transfected cells (Ctrl) is shown. Scale bar=10μm. n≥90 cells analyzed/sample, n=3 independent experiments.


G . Representative images of WIPI2 and OFD1 co - staining in KO - OFD1 cells transfected with empty vector ( Empty ) , 3xFLAG - OFD1 ( OFD1 ) or 3xFLAG - OFD1 ∆ LIR ( OFD1 ∆ LIR ) ( HBSS , 90min ) . Red , WIPI2 ; green , OFD1 ; blue , Hoechst labels nuclei . Scale bars : 10μm . On the right , quantification of WIPI2 puncta . ≥ 100 cells analyzed / sample , n = 4 independent experiments .

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G. Representative images of WIPI2 and OFD1 co-staining in KO-OFD1 cells transfected with empty vector (Empty), 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) (HBSS,90min). Red, WIPI2; green, OFD1; blue, Hoechst labels nuclei. Scale bars: 10μm. On the right, quantification of WIPI2 puncta. ≥100 cells analyzed/sample, n=4 independent experiments.


A . Western blot of LC3B in lymphoblasts of OFD type I patients and controls , either untreated ( - ) or exposed to Baf - A1 ( 10nM , 2h ) ( + ) . Cells were cultured in complete medium ( FM , top ) or under serum starvation for 4h ( STV , bottom ) . ACTIN and GAPDH were used as loading controls .

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A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.


B . ( Left ) Graphs show LC3B - II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium ( FM ) . ( Right ) Graphs show LC3B - II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h ( STV ) . Normalized LC3B - II levels are expressed as fold change versus untreated conditions ( - ) of control cells , n = 4 patients and 3 controls .

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B. (Left) Graphs show LC3B-II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium (FM). (Right) Graphs show LC3B-II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h (STV). Normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of control cells, n=4 patients and 3 controls.


C . Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients . ( Right ) Protein levels relative to ACTIN ( loading control ) are expressed as fold change versus controls , represented by the dashed line , n = 4 patients and 3 controls .

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C. Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients. (Right) Protein levels relative to ACTIN (loading control) are expressed as fold change versus controls, represented by the dashed line, n=4 patients and 3 controls.


A . Western blot of LC3B in kidney homogenates from leupeptin - treated ( 40mg / kg , 6h ) fasted Ofd1fl / y ; creKsp and control mice . ( Right ) LC3B - II protein levels normalized to Actin ( loading control ) are expressed as fold increase versus control mice , n = 4 mice / group .

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A. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1fl/y;creKsp and control mice. (Right) LC3B-II protein levels normalized to Actin (loading control) are expressed as fold increase versus control mice, n=4 mice/group.


B . Representative images of GFP - LC3 puncta ( autophagosomes , green ) in aquaporin2 - positive ( AQP2 , red ) renal distal tubules from P8 Ofd1fl / y ; creKsp ; GFP - LC3 and Ofd1fl / y ; GFP - LC3 mice . Scale bar = 10μm . Hoechst labels nuclei . Bar graphs on the left show quantitative analysis of GFP - LC3 puncta ; n = 4 mice / group for a total of 300 AQP2 positive nuclei / group analyzed .

B. Representative images of GFP-LC3 puncta (autophagosomes, green) in aquaporin2-positive (AQP2, red) renal distal tubules from P8 Ofd1fl/y;creKsp;GFP-LC3 and Ofd1fl/y;GFP-LC3 mice. Scale bar=10μm. Hoechst labels nuclei. Bar graphs on the left show quantitative analysis of GFP-LC3 puncta; n=4 mice/group for a total of 300 AQP2 positive nuclei/group analyzed.


C . Western blot of LC3B in kidney homogenates from leupeptin - treated ( 40mg / kg , 6h ) fasted Ofd1 - IND and control mice . ( Right ) LC3B - II protein levels relative to βTubulin ( loading control ) are expressed as fold increase compared with control mice , n = 5 mutant / 8 control mice .

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C. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1-IND and control mice. (Right) LC3B-II protein levels relative to βTubulin (loading control) are expressed as fold increase compared with control mice, n=5 mutant/8 control mice .


D . Western blot of Atg13 in Ofd1fl / y ; creKsp and control kidneys at pre - cystic ( P8 , left ) and cystic ( P22 , right ) stages . Gapdh was used as loading control . Atg13 protein levels are expressed as fold change compared with control mice ; n = 7 mutant / 5 control mice at P8 , n = 4mice / group at P22 .

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D. Western blot of Atg13 in Ofd1fl/y;creKsp and control kidneys at pre-cystic (P8, left) and cystic (P22, right) stages. Gapdh was used as loading control. Atg13 protein levels are expressed as fold change compared with control mice; n=7 mutant/5 control mice at P8, n= 4mice/group at P22.


E . Haematoxylin & eosin staining of kidney sections from Ofd1fl / y ; Atg7 + / + ; creKsp and Ofd1fl / y ; Atg7fl / fl ; creKsp mice . ( Right ) Graph shows fold change of the cysts number observed in Ofd1fl / y ; Atg7fl / fl ; creKsp animals compared with Ofd1fl / y ; Atg7 + / + ; creKsp ; n = 12 sections / animal were analyzed ; n = 6 mice / group . Scale bars = 1mm .

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E. Haematoxylin & eosin staining of kidney sections from Ofd1fl/y;Atg7+/+;creKsp and Ofd1fl/y;Atg7fl/fl;creKsp mice. (Right) Graph shows fold change of the cysts number observed in Ofd1fl/y;Atg7fl/fl;creKsp animals compared with Ofd1fl/y;Atg7+/+;creKsp; n=12 sections/animal were analyzed; n=6 mice/group. Scale bars=1mm.


F . Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl / y ; Atg7fl / fl ; creKsp , Ofd1fl / y ; Atg7 + / + ; creKsp and control mice at P45 . n = 8 mice per group .

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F. Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl/y;Atg7fl/fl;creKsp, Ofd1fl/y;Atg7+/+;creKsp and control mice at P45. n=8 mice per group.


Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group ( red line ) and EYFP group ( blue line ) in C48 / 80 model ( C ) . Heatmap illustration of fluorescence changes during each corresponding scratching train in GCaMP6s group . n = 8 mice / group . 2 trails for each are shown except for 1 mouse where 1 trial was performed ( D ) . * * * P < 0 . 001 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group .

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Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group (red line) and EYFP group (blue line) in C48/80 model (C). Heatmap illustration of fluorescence changes during each corresponding scratching train in GCaMP6s group. n = 8 mice/group. 2 trails for each are shown except for 1 mouse where 1 trial was performed (D). *** P < 0.001. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group.


J , Effect of chemogenetic inhibition ( J ) of dopaminergicA11 - SDH neurons on scratching behaviors in response to C48 / 80 ( left ) and CQ ( right ) stimuli . * * P < 0 . 01 , * * * P < 0 . 001 . Two - way ANOVA followed by Bonferroni ' s test . n = 6 - 8 mice / group .

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J, Effect of chemogenetic inhibition (J) of dopaminergicA11-SDH neurons on scratching behaviors in response to C48/80 (left) and CQ (right) stimuli. ** P < 0.01, *** P < 0.001. Two-way ANOVA followed by Bonferroni's test. n = 6-8 mice/group.


Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group ( red line ) and EYFP group ( blue line ) in DCP model ( B ) .

Average fluorescence signals aligned to the onset of scratching trains across the GCaMP6s group (red line) and EYFP group (blue line) in DCP model (B).


F , Effect of chemogenetic inhibition ( F ) of dopaminergicA11 - SDH neurons on DCP - induced scratching . * P < 0 . 05 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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F, Effect of chemogenetic inhibition (F) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


G Effect of chemogenetic activation ( G ) of dopaminergicA11 - SDH neurons on DCP - induced scratching . * P < 0 . 05 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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G Effect of chemogenetic activation (G) of dopaminergicA11-SDH neurons on DCP-induced scratching. * P < 0.05. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


A Effect of SCH23390 and sulpiride on scratching behaviors induced by C48 / 80 ( left ) and CQ ( right ) . * * * P < 0 . 001 . One - way ANOVA followed by Bonferroni ' s test . n = 7 - 8 mice / group .

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A Effect of SCH23390 and sulpiride on scratching behaviors induced by C48/80 (left) and CQ (right). *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 7-8 mice/group.


C Effect of SCH23390 and sulpiride on DCP - induced scratching behaviors . * P < 0 . 05 . One - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group .

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C Effect of SCH23390 and sulpiride on DCP-induced scratching behaviors. * P < 0.05. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group.


D The Drd1 mRNA level in the spinal cord after DCP or vehicle treatment . * P < 0 . 05 . Student ' s t - test . n = 8 mice / group .

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D The Drd1 mRNA level in the spinal cord after DCP or vehicle treatment. * P < 0.05. Student's t-test. n = 8 mice/group.


G Statistical curve shows the firing frequency evoked by step current injection into DRD1 + neurons that incubated with vehicle or SKF38393 . * * * P < 0 . 001 . Two - way ANOVA . n = 8 neurons / group .

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G Statistical curve shows the firing frequency evoked by step current injection into DRD1+ neurons that incubated with vehicle or SKF38393. *** P < 0.001. Two-way ANOVA. n = 8 neurons/group.


I Statistical shows the resting membrane potential in DRD1 + neurons incubated with vehicle or SKF38393 . * P < 0 . 05 . Paired Student ' s t - test . n = 8 neurons / group .

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I Statistical shows the resting membrane potential in DRD1+ neurons incubated with vehicle or SKF38393. * P < 0.05. Paired Student's t-test. n = 8 neurons/group.


K Representative traces from one spinal DRD1 + neuron show that blue light stimuli did not induce spontaneous firing ( holding potential , - 65 mV , lower panel ) but increased the existed firing of DRD1 + neuron ( holding potential , - 60 mV , upper panel ) . Arrows indicate blue light stimuli ( 2 - 3 mW / cm2 , 5 pulses , 2 ms duration , 20 Hz , repeated every 1 s ) . shows the change of firing frequency from individual DRD1 + neurons after blue light stimuli . * * P < 0 . 01 . Paired Student ' s t - test . n = 8 neurons / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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K Representative traces from one spinal DRD1+ neuron show that blue light stimuli did not induce spontaneous firing (holding potential, -65 mV, lower panel) but increased the existed firing of DRD1+ neuron (holding potential, -60 mV, upper panel). Arrows indicate blue light stimuli (2-3 mW/cm2, 5 pulses, 2 ms duration, 20 Hz, repeated every 1 s). shows the change of firing frequency from individual DRD1+ neurons after blue light stimuli. ** P < 0.01. Paired Student's t-test. n = 8 neurons/group. Data information: Bars represent mean values. Error bars indicate the SEM.


G Effect of chemogenetic activation of spinal DRD1 + neurons on spontaneous itch behaviors . * * * P < 0 . 001 . Student ' s t - test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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G Effect of chemogenetic activation of spinal DRD1+ neurons on spontaneous itch behaviors. *** P < 0.001. Student's t-test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


I Effect of chemogenetic inhibition ( left ) or activation ( right ) of spinal DRD1 + neurons on the DCP - induced chronic itch behaviors . * P < 0 . 05 , * * P < 0 . 01 . Two - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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I Effect of chemogenetic inhibition (left) or activation (right) of spinal DRD1+ neurons on the DCP-induced chronic itch behaviors. * P < 0.05, ** P < 0.01. Two-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


F - H Representative images show non - overlap of tdTomato with EGFP in the SDH of Drd1 - tdTomato ; Drd2 - EGFP mice . Scale bar , 100 μm .

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F-H Representative images show non-overlap of tdTomato with EGFP in the SDH of Drd1-tdTomato;Drd2-EGFP mice. Scale bar, 100 μm.


I Representative image shows the co - localization of NeuN with tdTomato . Scale bar , 50 μm . Inset scale bar , 25 μm .

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I Representative image shows the co-localization of NeuN with tdTomato. Scale bar, 50 μm. Inset scale bar, 25 μm.


J - M Representative images show the co - localization of NK1R ( J ) , CGRP ( K ) , IB4 ( L ) , and PKCγ ( M ) with tdTomato . Scale bar , 100 μm .

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J-M Representative images show the co-localization of NK1R (J), CGRP (K), IB4 (L), and PKCγ (M) with tdTomato. Scale bar, 100 μm.


N Co - localization of tdTomato fluorescence with vGluT2 - probe . Scale bar , 50 μm . O FISH double - staining of Drd1 - probe with vGluT2 - probe . White arrows and arrowheads show double - labeled neurons and vGluT2 single - labeled neurons , respectively . Scale bar , 10 μm . P Staining of PAX2 in Drd1 - tdTomato mice . Scale bar , 50 μm . Q Fluorescence of tdTomato and EGFP in the SDH of Drd1 - tdTomato ; Gad67 - EGFP mice . Scale bar , 50 μm .

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

N Co-localization of tdTomato fluorescence with vGluT2-probe. Scale bar, 50 μm. O FISH double-staining of Drd1-probe with vGluT2-probe. White arrows and arrowheads show double-labeled neurons and vGluT2 single-labeled neurons, respectively. Scale bar, 10 μm. P Staining of PAX2 in Drd1-tdTomato mice. Scale bar, 50 μm. Q Fluorescence of tdTomato and EGFP in the SDH of Drd1-tdTomato;Gad67-EGFP mice. Scale bar, 50 μm.


C Representative images show the reconstitution of pre - and post - mGRASP to form an intact GFP ( green ) , which mapped synaptic connectivity between spinal DRD1 + neurons ( red , as presynaptic constructs ) and GRPR + neurons ( upper panels ) . High - magnification images in the white frames were shown in lower panels . Scale bar , 20 μm in upper panel and 10 μm in lower panel .

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

C Representative images show the reconstitution of pre- and post-mGRASP to form an intact GFP (green), which mapped synaptic connectivity between spinal DRD1+ neurons (red, as presynaptic constructs) and GRPR+ neurons (upper panels). High-magnification images in the white frames were shown in lower panels. Scale bar, 20 μm in upper panel and 10 μm in lower panel.


G Representative traces of the whole - cell voltage - clamp show the blue light ( 470 nm , 2 ms ) - evoked EPSCs recorded in a GRPR + neuron before ( black ) and after 20 μM DNQX perfusion ( gray ) . H Representative traces show the effect of TTX alone ( purple ) and the combined presence of 4 - AP ( gray ) superfusion on the blue light - evoked EPSCs ( black ) recorded in a GRPR + neuron .

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

G Representative traces of the whole-cell voltage-clamp show the blue light (470 nm, 2 ms)-evoked EPSCs recorded in a GRPR+ neuron before (black) and after 20 μM DNQX perfusion (gray). H Representative traces show the effect of TTX alone (purple) and the combined presence of 4-AP (gray) superfusion on the blue light-evoked EPSCs (black) recorded in a GRPR+ neuron.


I Representative traces show the effect of TTX + 4 - AP ( gray ) on the blue light ( 470 nm , 2 ms ) - evoked EPSCs ( black ) recorded in a GRPR + neuron .

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

I Representative traces show the effect of TTX + 4-AP (gray) on the blue light (470 nm, 2 ms)-evoked EPSCs (black) recorded in a GRPR+ neuron.


D Single - cell RT - PCR shows the expression of Drd1 , Grp , NeuN , and Gapdh mRNA in tdTomato + neurons . N , negative control . * indicate Drd1 + Grp + neurons .

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

D Single-cell RT-PCR shows the expression of Drd1, Grp, NeuN, and Gapdh mRNA in tdTomato+ neurons. N, negative control. * indicate Drd1+Grp+ neurons.


I Effect of DNQX , RC - 3095 , or vehicle on the spontaneous scratching of mice with spinal DRD1 + neurons expressing mCherry or hM3Dq and activated by CNO . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA followed by Bonferroni ' s test . n = 8 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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

I Effect of DNQX, RC-3095, or vehicle on the spontaneous scratching of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO. ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 8 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


J Effect of DNQX , RC - 3095 , or vehicle on the scratching behaviors of mice with spinal DRD1 + neurons expressing mCherry or hM3Dq and activated by CNO in C48 / 80 model ( left ) or CQ model ( right ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA followed by Bonferroni ' s test . n = 6 mice / group . Data information : Bars represent mean values . Error bars indicate the SEM .

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

J Effect of DNQX, RC-3095, or vehicle on the scratching behaviors of mice with spinal DRD1+ neurons expressing mCherry or hM3Dq and activated by CNO in C48/80 model (left) or CQ model (right). * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA followed by Bonferroni's test. n = 6 mice/group. Data information: Bars represent mean values. Error bars indicate the SEM.


( C - G ' ' ) Lateral images of the cranial vessels in lyve1b : Kaede transgenic embryos with the CCV photoconverted at 36 hpf ( C ) and followed through to 48 hpf ( D ) and 60 hpf ( E ) , while in a separate larva the PHS has been photoconverted at 36 hpf ( F ) and followed to 48 hpf ( G ) . Unconverted vessels are shown in green ( C - G ) , while photoconverted vessels are shown in red ( C ' - G ' ) . The extent of lymphangioblast contribution from each source is further clarified by false colouring the overlap between red and green fluorescence ( purple ) , with the FLS demarcated ( dotted line ) from the adjacent primary veins ( C ' ' - G ' ' ) .

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

(C-G'') Lateral images of the cranial vessels in lyve1b:Kaede transgenic embryos with the CCV photoconverted at 36 hpf (C) and followed through to 48 hpf (D) and 60 hpf (E), while in a separate larva the PHS has been photoconverted at 36 hpf (F) and followed to 48 hpf (G). Unconverted vessels are shown in green (C-G), while photoconverted vessels are shown in red (C'-G'). The extent of lymphangioblast contribution from each source is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS demarcated (dotted line) from the adjacent primary veins (C''-G'').


( A - D ' ' ) Ventrolateral ( A , C , D ) and lateral ( B ) images of the cranial vessels in lyve1b : Kaede transgenic embryos with the dorsal ( A ) and ventral ( C ) portions of the VA - L photoconverted at 54 hpf and followed through to 72 hpf ( B ) and 78 hpf ( D ) . Unconverted vessels are shown in green ( A - D ) , while photoconverted vessels are shown in red ( A ' - D ' ) . The extent of lymphatic and arterial contribution from the VA - L to the LFL ( B ' ' ) or HA ( D ' ' ) is further clarified by false colouring the overlap between red and green fluorescence ( purple ) , with the FLS ( A ' ' , C ' ' ) and LFL ( B ' ' , D ' ' ) demarcated ( dotted line ) from the adjacent vessels . Schematic included for anatomical reference . Note , for all images , the overlap of red and green fluorescence in the eye ( labelled E ) is due to a combination of natural pigmentation and auto - fluorescence ; and is not indicative of photoconverted cells ( C ' ' , white arrowheads ) .

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

(A-D'') Ventrolateral (A, C, D) and lateral (B) images of the cranial vessels in lyve1b:Kaede transgenic embryos with the dorsal (A) and ventral (C) portions of the VA-L photoconverted at 54 hpf and followed through to 72 hpf (B) and 78 hpf (D). Unconverted vessels are shown in green (A-D), while photoconverted vessels are shown in red (A'-D'). The extent of lymphatic and arterial contribution from the VA-L to the LFL (B'') or HA (D'') is further clarified by false colouring the overlap between red and green fluorescence (purple), with the FLS (A'', C'') and LFL (B'', D'') demarcated (dotted line) from the adjacent vessels. Schematic included for anatomical reference. Note, for all images, the overlap of red and green fluorescence in the eye (labelled E) is due to a combination of natural pigmentation and auto-fluorescence; and is not indicative of photoconverted cells (C'', white arrowheads).


Lateral images of the CCV fixed at 36 hpf ( A ) and 42 hpf ( B ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) .

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

Lateral images of the CCV fixed at 36 hpf (A) and 42 hpf (B) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red).


Lateral images of the PHS at 36 hpf ( C ) , 42 hpf ( D ) and 48 hpf ( E ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . PHS Prox1 staining shown alone for all time points ( C ' - E ' ) with the FLS demarcated ( dotted line ) from the adjacent vessels , and the positions of the PHS - LPP and PHS - LPA indicated .

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

Lateral images of the PHS at 36 hpf (C), 42 hpf (D) and 48 hpf (E) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). PHS Prox1 staining shown alone for all time points (C'-E') with the FLS demarcated (dotted line) from the adjacent vessels, and the positions of the PHS-LPP and PHS-LPA indicated.


Lateral images of the VA - L at 36 hpf ( F ) , 42 hpf ( G ) and 48 hpf ( H ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . Note the Prox1 - postive ( green ) , GFP - negative structure near to the VA - L is a prox1 - expressing neuromast ( NM ) .

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

Lateral images of the VA-L at 36 hpf (F), 42 hpf (G) and 48 hpf (H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). Note the Prox1-postive (green), GFP-negative structure near to the VA-L is a prox1-expressing neuromast (NM).


( A - H ) Lateral images of the PHS fixed at 36 hpf ( A - B , E - F ) and 42 hpf ( C - D , G - H ) in lyve1b : EGFP fish that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . These show a decrease in the number of Prox1 - positive PHS ECs in the ccbe1 morpholino - injected ( B , D ) and sFLT4 mRNA - injected embryos ( F , H ) compared with control morpholino - injected ( A , C ) and uninjected embryos ( E , G ) . At 42 hpf , ccbe1 morpholino - injected embryos ( D ) and sFLT4 mRNA - injected embryos ( H ) lack the PHS - LP domains present in the control embryos ( C , G ; large arrow heads ) . ( I - J ) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino - injected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 17 ) and ccbe1 morpholino - injected embryos at 36 hpf ( n = 36 ) and 42 hpf ( n = 12 ) ( I ) or in uninjected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 21 ) and sFLT4 mRNA - injected embryos at 36 hpf ( n = 26 ) and 42 hpf ( n = 17 ) ( J ) . ( K ) Quantitation of the total number of PHS ECs in control morpholino - injected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 17 ) and ccbe1 morpholino - injected embryos at 36 hpf ( n = 36 ) and 42 hpf ( n = 12 ) or in uninjected embryos at 36 hpf ( n = 27 ) and 42 hpf ( n = 21 ) and sFLT4 mRNA - injected embryos at 36 hpf ( n = 26 ) and 42 hpf ( n = 17 )

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

(A-H) Lateral images of the PHS fixed at 36 hpf (A-B, E-F) and 42 hpf (C-D, G-H) in lyve1b:EGFP fish that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show a decrease in the number of Prox1-positive PHS ECs in the ccbe1 morpholino-injected (B, D) and sFLT4 mRNA-injected embryos (F, H) compared with control morpholino-injected (A, C) and uninjected embryos (E, G). At 42 hpf, ccbe1 morpholino-injected embryos (D) and sFLT4 mRNA-injected embryos (H) lack the PHS-LP domains present in the control embryos (C, G; large arrow heads). (I-J) Quantitation of the percentage of PHS ECs that are lymphatic progenitors relative to the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) (I) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17) (J). (K) Quantitation of the total number of PHS ECs in control morpholino-injected embryos at 36 hpf (n = 27) and 42 hpf (n = 17) and ccbe1 morpholino-injected embryos at 36 hpf (n = 36) and 42 hpf (n = 12) or in uninjected embryos at 36 hpf (n = 27) and 42 hpf (n = 21) and sFLT4 mRNA-injected embryos at 36 hpf (n = 26) and 42 hpf (n = 17)


( L - O ) Lateral images of the FLS ( L , N ; dotted line ) or dorsolateral CCV region from where the FLS normally sprouts ( M , O ; dotted line ) fixed at 36 hpf in lyve1b : EGFP embryos that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . These show no change in the number of Prox1 - positive lymphatic progenitors in the ccbe1 morpholino - injected ( M ) and sFLT4 mRNA - injected ( O ) embryos compared with control morpholino - injected ( L ) and uninjected ( N ) embryos . ( P ) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino - injected ( n = 26 ) , ccbe1 morpholino - injected ( n = 27 ) , uninjected ( n = 28 ) and sFLT4 mRNA - injected embryos ( n = 32 ) . ( Q ) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino ( n = 26 ) , ccbe1 morpholino ( n = 27 ) , or sFLT4 mRNA ( n = 32 ) .

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

(L-O) Lateral images of the FLS (L, N; dotted line) or dorsolateral CCV region from where the FLS normally sprouts (M, O; dotted line) fixed at 36 hpf in lyve1b:EGFP embryos that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show no change in the number of Prox1-positive lymphatic progenitors in the ccbe1 morpholino-injected (M) and sFLT4 mRNA-injected (O) embryos compared with control morpholino-injected (L) and uninjected (N) embryos. (P) Quantitation of the percentage of dorsolateral CCV ECs that are lymphatic progenitors relative to the total number of dorsolateral CCV ECs in control morpholino-injected (n = 26), ccbe1 morpholino-injected (n = 27), uninjected (n = 28) and sFLT4 mRNA-injected embryos (n = 32). (Q) Quantitation of the total number of dorsolateral region CCV ECs in 36 hpf embryos that were uninjected or injected with control morpholino (n = 26), ccbe1 morpholino (n = 27), or sFLT4 mRNA (n = 32).


( R - U ) Dorsolateral images of the VA - L fixed at 54 hpf in lyve1b : EGFP larva that have been fluorescently immunostained with anti - PROX1 ( green ) and anti - GFP ( red ) . These show that lymphatic progenitor formation still occurs within the VA - L in ccbe1 morpholino - injected ( S ) and sFLT4 mRNA - injected embryos ( U ) , with Prox1 expression resembling that of controls ( R , T ) .

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

(R-U) Dorsolateral images of the VA-L fixed at 54 hpf in lyve1b:EGFP larva that have been fluorescently immunostained with anti-PROX1 (green) and anti-GFP (red). These show that lymphatic progenitor formation still occurs within the VA-L in ccbe1 morpholino-injected (S) and sFLT4 mRNA-injected embryos (U), with Prox1 expression resembling that of controls (R, T).


( A - B ) Still images from Movie EV3 of early facial lymphatic development in a lyve1b : EGFP ( A ) , kdrl : nlsmCherry ( B ) compound transgenic from 42 - 51 . 5 hpf , with the CCV - L - derived leading tip cell ( green asterisk ) , the PHS - LP domains ( orange and yellow asterisk ) and the distal tip of the VA - L ( purple asterisk ) highlighted ( A ) . Between 42 and 46 hpf , the PHS - LPP ( orange outline ) cells divide ( dark blue parent cells , light blue daughter cells ) before sprouting as lymphangioblasts from the PHS and fusing with the tip ( pink cells ) of the CCV - derived FLS ( green outline ) at 48 hpf ( B ) . Cells ( red ) within the VA - L ( purple outline ) can also be seen migrating towards the now PHS - L - derived FLS tip by 48 hpf . The PHS - LPA is also shown ( yellow outline ) .

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

(A-B) Still images from Movie EV3 of early facial lymphatic development in a lyve1b:EGFP (A), kdrl:nlsmCherry (B) compound transgenic from 42-51.5 hpf, with the CCV-L-derived leading tip cell (green asterisk), the PHS-LP domains (orange and yellow asterisk) and the distal tip of the VA-L (purple asterisk) highlighted (A). Between 42 and 46 hpf, the PHS-LPP (orange outline) cells divide (dark blue parent cells, light blue daughter cells) before sprouting as lymphangioblasts from the PHS and fusing with the tip (pink cells) of the CCV-derived FLS (green outline) at 48 hpf (B). Cells (red) within the VA-L (purple outline) can also be seen migrating towards the now PHS-L-derived FLS tip by 48 hpf. The PHS-LPA is also shown (yellow outline).


( C ) Quantitation of leading lymphangioblast velocity ( µm / min ) before and after fusion with another lymphangioblast ( CCV - L - derived FLS tip ( pink cells ) to PHS - L ( B , blue cells ; n = 5 / 5 ) and PHS - L - derived FLS tip ( blue cells ) to VA - L ( F , red cells ; n = 5 / 5 ) ) or by the VA - L to the PHS - L - derived FLS tip ( B , n = 5 / 5 ) .

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

(C) Quantitation of leading lymphangioblast velocity (µm/min) before and after fusion with another lymphangioblast (CCV-L-derived FLS tip (pink cells) to PHS-L (B, blue cells; n = 5/5) and PHS-L-derived FLS tip (blue cells) to VA-L (F, red cells; n = 5/5)) or by the VA-L to the PHS-L-derived FLS tip (B, n = 5/5).


( D ) Cell migration tracks where each coloured track depicts the distance ( µm ) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV - L - derived FLS tip ( pink cells ) and the PHS - Ls ( B , blue cells ; n = 5 / 5 ) .

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

(D) Cell migration tracks where each coloured track depicts the distance (µm) travelled over 3 hours by the leading tip cell in a separate animal before and after fusion with the CCV-L-derived FLS tip (pink cells) and the PHS-Ls (B, blue cells; n = 5/5).


( E - F ) Still images from Movie EV4 of facial lymphatic development in a lyve1b : EGFP kdrl : nlsmCherry compound transgenic from 48 - 62 hpf showing that after the CCV - derived FLS fuses to the PHS - L , these take over as the leading tip cells ( orange asterisks ) , with one PHS - L migrating anteriorly to fuse with PHS - LPA ( yellow asterisk ) , and others migrating ventrally to fuse with the VA - L ( purple asterisk ) After the PHS - derived portion ( orange outline ) of the FLS ( green outline ) fuses to the VA - L ( purple outline ) , migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation ( dark blue and red parent cells , light blue and pink daughter cells ) before resuming migration anteriorly along the ventral base of the

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

(E-F) Still images from Movie EV4 of facial lymphatic development in a lyve1b:EGFP kdrl:nlsmCherry compound transgenic from 48-62 hpf showing that after the CCV-derived FLS fuses to the PHS-L, these take over as the leading tip cells (orange asterisks), with one PHS-L migrating anteriorly to fuse with PHS-LPA (yellow asterisk), and others migrating ventrally to fuse with the VA-L (purple asterisk) After the PHS-derived portion (orange outline) of the FLS (green outline) fuses to the VA-L (purple outline), migration of the entire facial lymphatic pauses to allow for lymphangioblast proliferation (dark blue and red parent cells, light blue and pink daughter cells) before resuming migration anteriorly along the ventral base of the eye