D Immunoblot analysis of retina lysates of 6 - 7 week old WT , NYKO , NOKO and NYOKO mice ( n = 3 ) . SDHA was used to control for gel loading .

O O O O O O O O O O O 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 Immunoblot analysis of retina lysates of 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3). SDHA was used to control for gel loading.


E Transverse semithin sections of spinal cords of 6 - 7 week old WT , NOKO and NYOKO mice . Sections were stained with toluidine blue . Orange arrows point to MDBs indicating degenerating neurons . Scale bars 25 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

E Transverse semithin sections of spinal cords of 6-7 week old WT, NOKO and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars 25 µm.


F MDBs in dorso - lateral tracts of 6 - 7 week old WT ( n = 3 ) , NOKO ( n = 3 ) , and NYOKO ( n = 3 ) mice . Data were analyzed using one - way ANOVA . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± 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 O O O O O O O O O O O O O O

F MDBs in dorso-lateral tracts of 6-7 week old WT (n = 3), NOKO (n = 3), and NYOKO (n = 3) mice. Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


G Walking beam test of 6 - 7 week old WT ( n = 6 ) , NOKO ( n = 5 ) and NYOKO ( n = 7 ) mice . Data were analyzed using one - way ANOVA . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , ns = not significant . Data are means ± 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 O O O O O O O O O O

G Walking beam test of 6-7 week old WT (n = 6), NOKO (n = 5) and NYOKO (n = 7) mice. Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.


H - I . Fibulin 7 immunostaining in 2 - month - vs . 2 - year - old tail section ( H ) and its intensity quantification per basal epidermal stem cell / basement membrane ( normalized to 2 - month - old ) ( I ) . Dotted box areas were enlarged in the lower panels . White arrows indicate fibulin 7 basement membrane staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I. Fibulin 7 immunostaining in 2-month- vs. 2-year-old tail section (H) and its intensity quantification per basal epidermal stem cell/basement membrane (normalized to 2-month-old) (I). Dotted box areas were enlarged in the lower panels. White arrows indicate fibulin 7 basement membrane staining.


D - E . Ki67 immunostaining in 1 - week - chase mice ( D ) and its quantitation ( E ) . White arrows show Ki67 and tdTomato double positive cells , which were counted and summarized in ( E ) . Scale bar : 20 μm . N = 5 WT / het and 3 KO mice .

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

D-E. Ki67 immunostaining in 1-week-chase mice (D) and its quantitation (E). White arrows show Ki67 and tdTomato double positive cells, which were counted and summarized in (E). Scale bar: 20 μm. N=5 WT/het and 3 KO mice.


F . Representative pictures from tail wounds of 1 - year - old Fbln7 WT and KO mice . G . Measurements of wound area over time in Fbln7 WT ( N = 5 ) and KO ( N = 7 ) mice .

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

F. Representative pictures from tail wounds of 1-year-old Fbln7 WT and KO mice. G. Measurements of wound area over time in Fbln7 WT (N=5) and KO (N=7) mice.


H - J . Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs . KO mice ( H ) and quantitation of the re - epithelialization length ( I ) and thickness at the healing front ( J ) . Scale bar : 200 μm . N = 3 WT and 5 KO mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J. Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs. KO mice (H) and quantitation of the re-epithelialization length (I) and thickness at the healing front (J). Scale bar: 200 μm. N=3 WT and 5 KO mice.


F - G . K31 and K14 immunostaining in tail sections of 2 - month - vs . 2 - year - old C57BL / 6J WT mice ( F ) and the quantification ( G ) . Dotted box areas are enlarged in the lower panels . Scale bar : 50 μm . Quantification of K14 intensity in the suprabasal area was normalized to basal area per cell . N = 4 ( 2 - 3 - month - old ) and N = 5 ( 2 - year - old ) C57BL / 6J mice ( 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

F-G. K31 and K14 immunostaining in tail sections of 2-month- vs. 2-year-old C57BL/6J WT mice (F) and the quantification (G). Dotted box areas are enlarged in the lower panels. Scale bar: 50 μm. Quantification of K14 intensity in the suprabasal area was normalized to basal area per cell. N=4 (2-3-month-old) and N=5 (2-year-old) C57BL/6J mice (G).


H - J . K31 and K14 immunostaining in tail sections of 1 - year - old Fbln7 WT vs . KO compared with 2 - month - old Fbln7 KO mice ( H ) and the quantitation ( I , J ) . Scale bar : 50 μm . N = 5 for both WT and KO of 2 - 3 - month - old mice ( I ) . N = 5 and N = 7 for 1 - year - old WT vs . KO , respectively ( J ) . Data information ( F - J )

H-J. K31 and K14 immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO compared with 2-month-old Fbln7 KO mice (H) and the quantitation (I, J). Scale bar: 50 μm. N=5 for both WT and KO of 2-3-month-old mice (I). N=5 and N=7 for 1-year-old WT vs. KO, respectively (J). Data information (F-J)


A . Collagen IV immunostaining in tail sections of 1 - year - old Fbln7 WT vs . KO . Dotted box regions are enlarged .

O O 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. Collagen IV immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO. Dotted box regions are enlarged.


B . Quantification of Collagen IV basement membrane intensity per cell in Fbln7 KO mice normalized to WT mice . N = 4 WT and N = 6 KO mice . Graphs show mean ± S . D . Mann - Whitney test . * * ; P < 0 . 01 , ns ; not significant .

B. Quantification of Collagen IV basement membrane intensity per cell in Fbln7 KO mice normalized to WT mice. N=4 WT and N=6 KO mice. Graphs show mean ± S.D. Mann-Whitney test. **; P<0.01, ns; not significant.


Distribution of RT shifts calculated with the - ΔL + ΔE method described in Appendix Figure S1 . Different transgenes in a mid - late - replicating region are compared : the IL2R reporter gene under the control of the βA - globin promoter ( βA pro ) containing an inactive ( i , N = 10 ) or an active origin ( ii , N = 8 ) flanked by two copies of FIV ( 2xFIV ) , the blasticidin resistance gene ( BsR ) under the control of the β - actin promoter ( β - act pro ) ( iii , N = 7 ) or a combination of these two transgenes ( iv , N = 8 ) . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

Distribution of RT shifts calculated with the -ΔL+ΔE method described in Appendix Figure S1. Different transgenes in a mid-late-replicating region are compared: the IL2R reporter gene under the control of the βA-globin promoter (βA pro) containing an inactive (i, N=10) or an active origin (ii, N=8) flanked by two copies of FIV( 2xFIV), the blasticidin resistance gene (BsR) under the control of the β-actin promoter (β-act pro) (iii, N=7) or a combination of these two transgenes (iv, N=8). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (ns, not significant; **p<0.01; ***p<0.001).


A . UCSC genome browser visualization of the mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 71 , 000 , 000 - 74 , 100 , 000 bp , 3 . 1 Mb ; galGal5 ) . RT weighted average ( WA ) values for the wt and the βA - globin + β - actin cell lines are shown . Early - replicated regions ( E ) are represented in orange and late replicating regions ( L ) in blue . Annotated genes ( Ref Seq genes ) are represented below . The mid - late insertion site is indicated with a red arrow and dotted line .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:71,000,000-74,100,000 bp, 3.1 Mb; galGal5). RT weighted average (WA) values for the wt and the βA-globin+β-actin cell lines are shown. Early-replicated regions (E) are represented in orange and late replicating regions (L) in blue. Annotated genes (Ref Seq genes) are represented below. The mid-late insertion site is indicated with a red arrow and dotted line.


B . UCSC genome browser visualization of the mid - late insertion site of chromosome 1 ( genomic positions : chr1 : 72 , 450 , 000 - 72 , 650 , 000 bp ; 200 kb ; galGal5 ) . Tracks of nascent strands ( NS ) enrichments in the four S phase fractions were represented separately ( S1 to S4 ) for the wt and the 2x ( βA - globin + β - actin ) cell lines . NS enriched and depleted regions for each fraction are represented in purple and blue respectively . Single reads form SNS aligned and track of replication origins ( Ori peaks ) determined in ( Massip et al , 2019 ) are reported in between . The three mid - late insertion sites 1 , 2 and 3 are indicated with red arrows . Annotated genes and CpG Islands are shown below . Initiation zones ( IZ ) and termination zones ( TZ ) are reported .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UCSC genome browser visualization of the mid-late insertion site of chromosome 1 (genomic positions: chr1:72,450,000-72,650,000 bp; 200 kb; galGal5). Tracks of nascent strands (NS) enrichments in the four S phase fractions were represented separately (S1 to S4) for the wt and the 2x(βA-globin+β-actin) cell lines. NS enriched and depleted regions for each fraction are represented in purple and blue respectively. Single reads form SNS aligned and track of replication origins (Ori peaks) determined in (Massip et al, 2019) are reported in between. The three mid-late insertion sites 1, 2 and 3 are indicated with red arrows. Annotated genes and CpG Islands are shown below. Initiation zones (IZ) and termination zones (TZ) are reported.


A . Distribution of - ΔL + ΔE values for clonal cell lines containing one advanced replicon ( βA - globin + β - actin ) inserted at site 1 ( i , N = 8 ) or 3 ( iii , N = 3 ) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA - globin promoter / origin linked to a 1 . 6 kb fragment of human chromosome 7 ( h . K7 ) inserted at site 2 , either on the same chromosome as 1 and 3 ( 1 + 2 + 3 ) , or on the other chromosome ( 1 + 3 ) ( ii , N = 5 ) . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of -ΔL+ΔE values for clonal cell lines containing one advanced replicon (βA-globin+β-actin) inserted at site 1 (i, N=8) or 3 (iii, N=3) or at both sites on the same chromosome with one GFP reporter construct composed of the GFP reporter gene under the control of the βA-globin promoter/origin linked to a 1.6 kb fragment of human chromosome 7 (h.K7) inserted at site 2, either on the same chromosome as 1 and 3 (1+2+3), or on the other chromosome (1+3) (ii, N=5). Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


C . Interaction profiles of allele 1 + 3 / 2 with chr1 for combined Hi - C libraries : distance - corrected interactions , i . e . observed / expected ( OE ) , binned at 500 kb with corresponding compartment ( EV1 ) tracks . Red arrows represent the loci that show preferred interactions with allele 1 + 3 and that coincide with A compartment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Interaction profiles of allele 1+3/2 with chr1 for combined Hi-C libraries: distance-corrected interactions, i.e. observed/expected (OE), binned at 500 kb with corresponding compartment ( EV1) tracks. Red arrows represent the loci that show preferred interactions with allele 1+3 and that coincide with A compartment.


D . OE interactions of allele 1 + 3 ( orange rectangles ) and allele 2 ( blue rectangles ) averaged over A and B compartments for p and q - arms at 100kb bin size . A random compartment status permutation test was made and then we calculated the one - sided p - value for the observed value of ' difference allele 1 + 3 / 2 A - B ' . Values corresponding to OE interactions of allele 1 + 3 and allele 2 averaged over shuffled A ( red circles , N = 1000 ) and B compartments ( blue circles , N = 1000 ) for p and q - arms . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

D. OE interactions of allele 1+3 (orange rectangles) and allele 2 (blue rectangles) averaged over A and B compartments for p and q-arms at 100kb bin size. A random compartment status permutation test was made and then we calculated the one-sided p-value for the observed value of 'difference allele 1+3 / 2 A - B'. Values corresponding to OE interactions of allele 1+3 and allele 2 averaged over shuffled A (red circles, N=1000) and B compartments (blue circles, N=1000) for p and q-arms. ns, not significant; **p<0.01;***p<0.001.


E . Scatterplot of density of interactions between allele 1 + 3 and allele 2 with chromosomes larger than 5 , 000 , 000 bp .

E. Scatterplot of density of interactions between allele 1+3 and allele 2 with chromosomes larger than 5,000,000 bp.


A . Distribution of - ΔL + ΔE values for clonal cell lines containing one β - actin construct at site 1 ( i , N = 7 ) or at site 1 and 3 ( ii , N = 5 ) , on the same chromosome . Data information : Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

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

A. Distribution of -ΔL+ΔE values for clonal cell lines containing one β-actin construct at site 1 (i, N=7) or at site 1 and 3 (ii, N=5), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


B . Distribution of - ΔL + ΔE values for clonal cell lines containing one βA - globin construct at site 1 ( i , N = 8 ) or at site 1 and 3 ( ii , N = 6 ) , on the same chromosome . Data information : Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Rectangle edges correspond to the 0 . 25 and 0 . 75 quartiles , the thick black lines represent the median , the white triangles represent the mean and the whiskers extend to the smallest and largest - ΔL + ΔE values . Statistical analysis was performed with Wilcoxon nonparametric two - tailed tests ( * * p < 0 . 01 ) .

B. Distribution of -ΔL+ΔE values for clonal cell lines containing one βA-globin construct at site 1 (i, N=8) or at site 1 and 3 (ii, N=6), on the same chromosome. Data information: Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Rectangle edges correspond to the 0.25 and 0.75 quartiles, the thick black lines represent the median, the white triangles represent the mean and the whiskers extend to the smallest and largest -ΔL+ΔE values. Statistical analysis was performed with Wilcoxon nonparametric two-tailed tests (**p<0.01).


C . After 1 , 8 , 24 and 48 h of 4 - hydroxytamoxifen treatment , genomic DNA was extracted and quantified by semi - quantitative PCR ( see Appendix Figure S6 ) . Error bars correspond to the standard deviation for PCR duplicates . The percentages of cells with the large 1 + 3 and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) or β - actin ( 1 + 3 ) excision at each time point are shown for different cell lines .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. After 1, 8, 24 and 48 h of 4-hydroxytamoxifen treatment, genomic DNA was extracted and quantified by semi-quantitative PCR (see Appendix Figure S6). Error bars correspond to the standard deviation for PCR duplicates. The percentages of cells with the large 1+3 and loxP_LE (1) + βA-globin+β-actin (3) or β-actin (1+3) excision at each time point are shown for different cell lines.


A , B . Clones 1 + 2 + 3 , 1 + 3 ( A ) and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) ( B ) were used to assess chromatin accessibility within the transgenes . The positions of the amplicons used for quantification in each shifting construct individually ( thick black lines ; 5 ' 2xFIV - 1 or - 3 , β - actin pro - 1 or - 3 , BsR gene , PuroR gene , LoxP site ) , in both shifting constructs ( βA pro 1 and 2 , IL2R gene , 3 ' 2xFIV1 - 3 ) or in the GFP reporter construct ( βA - GFP , GFP gene , h . K7 ) are shown . The insertion sites on the wt chromosome ( insertion sites 1 , 2 and 3 ) and a genomic region located 5 kb downstream from the insertion site ( insertion site + 5kb ) were used as controls for the targeted genomic region . The endogenous active MED14 promoter was analyzed as a control . Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively . Black vertical bars represent insertion sites . Quantifications by real - time qPCR of total chromatin ( input ) extracted from two 1 + 2 + 3 , 1 + 3 and loxP _ LE ( 1 ) + βA - globin + β - actin ( 3 ) clonal cell lines , after digestion with micrococcal nuclease ( MNAse , 2 . 5 U , 10 U , 40 U , 160 U / mL ) and size selection were shown . Error bars indicate the standard deviation for qPCR triplicates , for two independent clones . Data are presented as total chromatin input relative to the condensed genomic regions ( cond1 and cond2 ) used for the normalization .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Clones 1+2+3, 1+3 (A) and loxP_LE (1) + βA-globin+β-actin (3) (B) were used to assess chromatin accessibility within the transgenes. The positions of the amplicons used for quantification in each shifting construct individually (thick black lines; 5' 2xFIV-1 or -3, β-actin pro-1 or -3, BsR gene, PuroR gene, LoxP site), in both shifting constructs (βA pro 1 and 2, IL2R gene, 3' 2xFIV1-3) or in the GFP reporter construct (βA-GFP, GFP gene, h.K7) are shown. The insertion sites on the wt chromosome (insertion sites 1, 2 and 3) and a genomic region located 5 kb downstream from the insertion site (insertion site +5kb) were used as controls for the targeted genomic region. The endogenous active MED14 promoter was analyzed as a control. Blue and black triangles represent reactive loxP sites and recombined inactive loxP sites respectively. Black vertical bars represent insertion sites. Quantifications by real-time qPCR of total chromatin (input) extracted from two 1+2+3, 1+3 and loxP_LE (1)+ βA-globin+β-actin (3) clonal cell lines, after digestion with micrococcal nuclease (MNAse, 2.5 U, 10 U, 40 U, 160 U/mL) and size selection were shown. Error bars indicate the standard deviation for qPCR triplicates, for two independent clones. Data are presented as total chromatin input relative to the condensed genomic regions (cond1 and cond2) used for the normalization.


A , B . UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites ( genomic positions : chr1 : 69 , 000 , 000 - 74 , 200 , 000 ; 5 . 2 Mb ( A ) and chr1 : 175 , 300 , 000 - 180 , 500 , 000 ; 5 . 2 Mb ( B ) ; galGal5 ) . RT weighted average ( WA ) values obtained after cells sorting into four S - phase fractions , BrdU pulse - labeled nascent strands ( NS ) immunoprecipitation and sequencing are shown . Early - replicated regions are represented in orange and late - replicating regions in blue . Below , track of eigenvector 1 values corresponding to A / B compartments at 100 kb resolution after Hi - C analysis in wt cells , track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) and annotated genes are reported . The three sites chosen for insertion are indicated with red arrows and dotted lines ( mid - late , late 1 ( A ) and late 2 ( B ) insertion ) . At the bottom , an enlargement of late insertion sites ( chr1 : 69 , 500 , 000 - 71 , 500 , 000 ( A ) , chr1 : 177 , 000 , 000 - 179 , 000 , 000 ( B ) ; galGal5 ) , displaying genes , mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in ( Massip et al , 2019 ) ( Ori peaks ) is shown . Tracks of RefSeq genes are represented in blue , tracks of Ensembl genes in red , tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black . Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UCSC genome browser visualization of two parts of chromosome 1 containing the insertion sites (genomic positions: chr1:69,000,000-74,200,000; 5.2 Mb (A) and chr1: 175,300,000-180,500,000; 5.2 Mb (B); galGal5). RT weighted average (WA) values obtained after cells sorting into four S-phase fractions, BrdU pulse-labeled nascent strands (NS) immunoprecipitation and sequencing are shown. Early-replicated regions are represented in orange and late-replicating regions in blue. Below, track of eigenvector 1 values corresponding to A/B compartments at 100 kb resolution after Hi-C analysis in wt cells, track of replication origins determined in (Massip et al, 2019) (Ori peaks) and annotated genes are reported. The three sites chosen for insertion are indicated with red arrows and dotted lines (mid-late, late 1 (A) and late 2 (B) insertion). At the bottom, an enlargement of late insertion sites (chr1: 69,500,000-71,500,000 (A), chr1: 177,000,000-179,000,000 (B); galGal5), displaying genes, mRNAs and ESTs for the chicken and all other species together with track of replication origins determined in (Massip et al, 2019) (Ori peaks) is shown. Tracks of RefSeq genes are represented in blue, tracks of Ensembl genes in red, tracks of AUGUSTUS ab initio gene predictions and TransMap alignements in green and tracks of mRNAs and ESTs in black. Insertion sites are indicated with red dotted lines and black dotted lines determine the borders of the enlarged regions.


A , B . RT profiles of chromosomal alleles following the targeted integration of a βA - globin + β - actin construct into two late - replicating loci , 1 ( A ) and 2 ( B ) in two or three clonal cell lines respectively . Nascent strands ( NS ) were quantified by real - time qPCR in four S phase fractions . Specific primer pairs determine the RT profile for the modified allele ( With ) , the wt allele ( Without ) and both alleles ( Both ) . The endogenous β - globin locus was analyzed as an early - replicated control . The modified ( Mod ) and wild - type ( wt ) alleles are shown . Difference - ΔL + ΔE values calculated at the target site following transgene integration are indicated ( see Appendix Figure S1 ) . Error bars correspond to the standard deviation for qPCR duplicates . The black vertical bars represent the precise insertion position .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT profiles of chromosomal alleles following the targeted integration of a βA-globin+β-actin construct into two late-replicating loci, 1 (A) and 2 (B) in two or three clonal cell lines respectively. Nascent strands (NS) were quantified by real-time qPCR in four S phase fractions. Specific primer pairs determine the RT profile for the modified allele (With), the wt allele (Without) and both alleles (Both). The endogenous β-globin locus was analyzed as an early-replicated control. The modified (Mod) and wild-type (wt) alleles are shown. Difference -ΔL+ΔE values calculated at the target site following transgene integration are indicated (see Appendix Figure S1). Error bars correspond to the standard deviation for qPCR duplicates. The black vertical bars represent the precise insertion position.


( A ) ADAR2 is most strongly expressed in vascular tissues . Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues . Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ADAR2 is most strongly expressed in vascular tissues. Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues. Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( B ) FLNA gene expression in few representative human tissues from > 500 different donors using GTEx data . Tibial artery , colon and esophagus show highest gene expression similar to ADAR2 expression . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FLNA gene expression in few representative human tissues from >500 different donors using GTEx data. Tibial artery, colon and esophagus show highest gene expression similar to ADAR2 expression. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( C ) Bar graph shows FLNA RNA editing ( % ) among few representative human tissues . Note very high editing levels in the arterial system ( red bars ) .

O O O O O O O O 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) Bar graph shows FLNA RNA editing (%) among few representative human tissues. Note very high editing levels in the arterial system (red bars).


( D ) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients . FLNA marked in the graph is one significant discriminator . Y axis is the - log 10 of the p value for the difference between healthy and sick . The colors ( red vs green ) reflect the threshold cutoffs randomly assigned to distinguish the sites , which demonstrate large , highly significant differences between the two groups . Fold change cutoff of 0 . 9 was used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients. FLNA marked in the graph is one significant discriminator. Y axis is the -log 10 of the p value for the difference between healthy and sick. The colors (red vs green) reflect the threshold cutoffs randomly assigned to distinguish the sites, which demonstrate large, highly significant differences between the two groups. Fold change cutoff of 0.9 was used.


( E ) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta . At least 4 control and 7 diseased human donors were used in each case . P value < 0 . 05 measured by t test was considered significant .

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

(E) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta. At least 4 control and 7 diseased human donors were used in each case. P value <0.05 measured by t test was considered significant.


( A ) Scheme showing the FLNA wild - type ( wt ) allele , the targeting vector and the FLNAΔECS neo - allele . The loxP flanked PGK - neo cassette in the targeting vector replaced the editing complementary sequence ( ECS ) using homologous recombination , which was then deleted using Cre recombinase . Right , southern blotting analysis screened for positive clones shown . The positions of loxP sites , restriction enzyme ( EcoRI ) and Southern blotting probe are also 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

(A) Scheme showing the FLNA wild-type (wt) allele, the targeting vector and the FLNAΔECS neo-allele. The loxP flanked PGK-neo cassette in the targeting vector replaced the editing complementary sequence (ECS) using homologous recombination, which was then deleted using Cre recombinase. Right, southern blotting analysis screened for positive clones shown. The positions of loxP sites, restriction enzyme (EcoRI) and Southern blotting probe are also indicated.


( B ) Sequencing electropherograms show the average FLNA editing levels in wt and FLNAΔECS tissues ( Stomach , colon , heart , lung and tibial artery ) . Editing levels were checked in tissues from 3 independent mice and the value below the chromatogram depict the average value of 3 replicates . Data information : Arrows indicate the FLNA editing site .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Sequencing electropherograms show the average FLNA editing levels in wt and FLNAΔECS tissues (Stomach, colon, heart, lung and tibial artery). Editing levels were checked in tissues from 3 independent mice and the value below the chromatogram depict the average value of 3 replicates. Data information: Arrows indicate the FLNA editing site.


( C ) FLNA mRNA expression levels measured by qPCR in wt and FLNAΔECS colon tissue showed no difference . Data information : data shown as mean ± SD from three independent experiments . * * P < 0 .

O O O O O O O O O O O O O O O O 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) FLNA mRNA expression levels measured by qPCR in wt and FLNAΔECS colon tissue showed no difference. Data information: data shown as mean ± SD from three independent experiments. ** P<0.05


( D ) FLNA protein levels measured by western blotting are identical in wt and FLNAΔECS stomach tissue . Y - axis represents FLNA expression normalized to tubulin levels . Data information : data shown as mean ± SD from three independent experiments . * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FLNA protein levels measured by western blotting are identical in wt and FLNAΔECS stomach tissue. Y-axis represents FLNA expression normalized to tubulin levels. Data information: data shown as mean ± SD from three independent experiments. ** P<0.05


( E ) FLNA editing levels ( % ) in wt whole dorsal aorta , tunica media and tunica adventitia . Tunica media consisting of smooth muscle cells show the highest FLNA RNA editing . Data information : Arrows indicate the FLNA editing site . data shown as mean ± SD from three independent experiments . * * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FLNA editing levels (%) in wt whole dorsal aorta, tunica media and tunica adventitia. Tunica media consisting of smooth muscle cells show the highest FLNA RNA editing. Data information: Arrows indicate the FLNA editing site. data shown as mean ± SD from three independent experiments. ** P<0.05


A , B ) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to ( A ) hypercontraction and ( B ) increased contraction force in FLNAΔECS aortae in myograph chambers . Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae . For each condition , 10 - 12 aortic rings from at least 4 wild - type ( wt ) and 4 FLNAΔECS mice were used . Data shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test )

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

A,B) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to (A) hypercontraction and (B) increased contraction force in FLNAΔECS aortae in myograph chambers. Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae. For each condition, 10-12 aortic rings from at least 4 wild-type (wt) and 4 FLNAΔECS mice were used. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001 (Student's t test)


( C ) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt ( red ) and FLNAΔECS ( green ) primary vascular smooth muscle cells ( vSMC ) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619 . Data shown as mean ± SD from three 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 B-CONTROLLED_VAR O O O O O O O O O O O O

(C) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt (red) and FLNAΔECS (green) primary vascular smooth muscle cells (vSMC) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619. Data shown as mean ± SD from three independent experiments.


( D ) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619 . Data shown as mean ± SD from three independent experiments . P value < 0 . 05 ( Student ' s t test ) was considered significant . The difference between wt and FLNAΔECS was significant at every concentration of U46619 .

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

(D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.


( A ) Myosin light chain phosphorylation detected by a phospho - specific antibody ( pMLC ) and normalized to total myosin light chain ( MLC ) . Tubulin served as a loading control . Quantification shows a significant increase in pMLC in FLNAΔECS vSMCs . Data information data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Myosin light chain phosphorylation detected by a phospho-specific antibody (pMLC) and normalized to total myosin light chain (MLC). Tubulin served as a loading control. Quantification shows a significant increase in pMLC in FLNAΔECS vSMCs. Data information data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( B ) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated ( GTP ) bound RhoA was compared between the 2 genotypes . FLNAΔECS vSMCs show more GTP - loaded RhoA than wt vSMCs . Tubulin was used as a loading control . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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 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

(B) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated (GTP) bound RhoA was compared between the 2 genotypes. FLNAΔECS vSMCs show more GTP-loaded RhoA than wt vSMCs. Tubulin was used as a loading control. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( C ) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase ( pMYPT1 ) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor - specific MYPT1 antibody . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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

(C) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase (pMYPT1) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor-specific MYPT1 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


( D ) CPI17 phosphorylation levels were measured in wt and FLNAΔECS vSMCs in untreated cells and in the presence of ROCK and PKC inhibitor using pCPI17 antibody . Data information : data shown as mean ± SD from at least three independent experiments . * * P < 0 . 05 ( Student ' s t test )

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

(D) CPI17 phosphorylation levels were measured in wt and FLNAΔECS vSMCs in untreated cells and in the presence of ROCK and PKC inhibitor using pCPI17 antibody. Data information: data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)


A - C ) Telemetry measurements were done on mice continuously for 72 hrs and reading was recorded every minute to analyse blood pressure in wt and FLNAΔECS mice . Scatter plots show diastolic blood pressure over 72 hrs ( A ) , diastolic blood pressure during inactive phase ( B ) and diastolic pressure during active phase ( C ) . Note : Significant increase in diastolic blood pressure in FLNAΔECS mice during the inactive phase . 9 mice ( 5 males marked as square + 4 females marked as round ) were analyzed for each genotype . Data shown as mean ± SEM . P value < 0 . 05 ( Student ' s t test ) was considered significant . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Telemetry measurements were done on mice continuously for 72 hrs and reading was recorded every minute to analyse blood pressure in wt and FLNAΔECS mice. Scatter plots show diastolic blood pressure over 72 hrs (A), diastolic blood pressure during inactive phase (B) and diastolic pressure during active phase (C). Note: Significant increase in diastolic blood pressure in FLNAΔECS mice during the inactive phase. 9 mice (5 males marked as square + 4 females marked as round) were analyzed for each genotype. Data shown as mean ± SEM. P value <0.05 (Student's t test) was considered significant. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.


( A ) Dorsal aortae were fixed , cross - sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel . 6 independent mice ( 5 - 6 months ) of each genotype were used for the analysis . Scale bar : 50 μm . ( B ) Plot shows individual data points ( black - wt , red - mutant ) of adventitial area in both the genotypes . Measurements show increased adventitial area in FLNAΔECS aortae . P value < 0 . 05 was considered significant ( Student ' s t test )

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 O O O O O

(A) Dorsal aortae were fixed, cross-sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel. 6 independent mice (5-6 months) of each genotype were used for the analysis. Scale bar: 50 μm. (B) Plot shows individual data points (black-wt, red-mutant) of adventitial area in both the genotypes. Measurements show increased adventitial area in FLNAΔECS aortae. P value <0.05 was considered significant (Student's t test)


( C ) Representative heart sections of a 5 - 6 months old mice stained with Masson Trichrome shows increased collagen ( blue ) around coronary vessels ( marked by arrowheads ) Scale bar : 50 μm . ( D ) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts . Four sections from each heart were measured . Four mice analyzed for each genotype . P value < 0 . 05 considered significant . ( mixed model approach )

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

(C) Representative heart sections of a 5-6 months old mice stained with Masson Trichrome shows increased collagen (blue) around coronary vessels (marked by arrowheads) Scale bar: 50 μm. (D) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts. Four sections from each heart were measured. Four mice analyzed for each genotype. P value <0.05 considered significant. (mixed model approach)


( E ) Representative longitudinal heart sections stained with H & E staining ( RV : right ventricle , LV : left ventricle ) . Black lines represent thickness of interventricular ( IVS ) and lateral left ventricular wall ( LLVH ) . Scale bar : 1mm . ( F ) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts . Six male mice ( 6months old ) analyzed for each genotype . P value < 0 . 05 was considered significant . ( Student ' s t test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 longitudinal heart sections stained with H&E staining (RV: right ventricle, LV: left ventricle). Black lines represent thickness of interventricular (IVS) and lateral left ventricular wall (LLVH). Scale bar: 1mm. (F) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts. Six male mice (6months old) analyzed for each genotype. P value <0.05 was considered significant. (Student's t test)


( G ) Heart cross - sections were stained with FITC - WGA ( Wheat germ agglutinin ) to visualize cell membranes of cardiomyocytes . Scale bar : 20 μm . ( H ) Respective cell areas measured by Image J software and represented by box plots for each animal ( six per genotype ) . Boxes represent the 25th and the 75th percentile with median represented by the black line in the box . The whiskers depict the minimum and the maximum value . P value < 0 . 05 ( mixed model approach ) was considered significant .

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

(G) Heart cross-sections were stained with FITC-WGA (Wheat germ agglutinin) to visualize cell membranes of cardiomyocytes. Scale bar: 20 μm. (H) Respective cell areas measured by Image J software and represented by box plots for each animal (six per genotype). Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. P value <0.05 (mixed model approach) was considered significant.


( A ) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice ( shades of grey ) and 5 FLNAΔECS mice ( shades of red ) . The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort . Aortic velocity waveform represents velocity during different stages of cardiac cycle . A significant reduction of velocity was observed at 40ms in the systolic cycle .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice (shades of grey) and 5 FLNAΔECS mice (shades of red). The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort. Aortic velocity waveform represents velocity during different stages of cardiac cycle. A significant reduction of velocity was observed at 40ms in the systolic cycle.


( B ) Representative wt ( top ) and FLNAΔECS ( below ) mouse showing the blood flow just above the aortic valve . Note : Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring . The red circle represents the time window at which the images are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative wt (top) and FLNAΔECS (below) mouse showing the blood flow just above the aortic valve. Note: Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring. The red circle represents the time window at which the images are shown.


( B ) High magnification view of boxed regions in ( A ) pseudo - colored to show actin depletion in anaphase . Scale bar - 1 µm .

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

(B) High magnification view of boxed regions in (A) pseudo-colored to show actin depletion in anaphase. Scale bar -1 µm.


( F ) Stills from time - lapse sequence of representative Hela cells expressing mNeon - Green Aurora B and Cell Mask to label cell membrane , exiting mitosis , showing the dynamic re - localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment ( arrows top ) . With knock - down of RACGAP1 , Aurora B still re - localises from the DNA to the microtubules and furrow in the midzone in early anaphase ( arrows bottom ) , but the microtubule localization is lost at later stages ( asterisk bottom ) . Scale bar - 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stills from time-lapse sequence of representative Hela cells expressing mNeon-Green Aurora B and Cell Mask to label cell membrane, exiting mitosis, showing the dynamic re-localization of Aurora B from DNA to the overlapping microtubules and cleavage furrow with control siRNA treatment (arrows top). With knock-down of RACGAP1, Aurora B still re-localises from the DNA to the microtubules and furrow in the midzone in early anaphase (arrows bottom), but the microtubule localization is lost at later stages (asterisk bottom). Scale bar - 10 µm.


( E ) Stills of representative Hela cells expressing Lifeact - GFP and H2B - mCherry at 10 mins following forced mitotic exit with siControl , siRACGAP1 , DMSO and 2µM ZM447439 treatment . Control siRNA , RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA , while ZM447439 treated cells fail to clear actin , quantified in ( F ) . Note , failure in actin accumulation in RACGAP1 depleted cell , asterisk . Scale bar - 10

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

(E) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry at 10 mins following forced mitotic exit with siControl, siRACGAP1, DMSO and 2µM ZM447439 treatment. Control siRNA, RACGAP1 siRNA and DMSO treated cells show clearance of actin from the cortex close to the DNA, while ZM447439 treated cells fail to clear actin, quantified in (F). Note, failure in actin accumulation in RACGAP1 depleted cell, asterisk. Scale bar - 10 µm


( F ) Quantification of actin intensity across the perimeter of the cells based on distance from DNA , with 5 and 6 being closest to DNA and 1 and 10 furthest away , as in cartoon ( B ) . Graph shows decrease in actin intensity closer to the DNA in siControl ( n = 26 ) , siRACGAP1 ( n = 37 ) and Control DMSO treated cells ( n = 38 cells ) , seen by levels lower than 1 ( dotted red line ) , while this decrease is absent in ZM447439 treated cells ( n = 42 cells ) . Data presented as mean ± SD .

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

(F) Quantification of actin intensity across the perimeter of the cells based on distance from DNA, with 5 and 6 being closest to DNA and 1 and 10 furthest away, as in cartoon (B). Graph shows decrease in actin intensity closer to the DNA in siControl (n=26), siRACGAP1 (n=37) and Control DMSO treated cells (n=38 cells), seen by levels lower than 1 (dotted red line), while this decrease is absent in ZM447439 treated cells (n=42 cells). Data presented as mean ± SD.


( B ) Stills from time - lapse sequence of the basal most region of Hela cells expressing Lifeact - GFP and H2B - mCherry forced to undergo flat monopolar mitotic exit . While actin is cleared underneath the DNA in control DMSO treated cells , it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit . Dotted mask shows position of DNA in actin channel . Scale bar - 10µm .

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

(B) Stills from time-lapse sequence of the basal most region of Hela cells expressing Lifeact-GFP and H2B-mCherry forced to undergo flat monopolar mitotic exit. While actin is cleared underneath the DNA in control DMSO treated cells, it fails to be cleared around the DNA when cells are treated with Aurora B inhibitor ZM447439 during mitotic exit. Dotted mask shows position of DNA in actin channel. Scale bar -10µm.


( C ) Quantification of clearance of actin underneath the DNA for still shown in ( B ) . While DNA under control cell starts clearing actin within 10 mins , Aurora B inhibited cells fail to do so .

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

(C) Quantification of clearance of actin underneath the DNA for still shown in (B). While DNA under control cell starts clearing actin within 10 mins, Aurora B inhibited cells fail to do so.


( D ) Still images of representative Hela cells expressing Lifeact - GFP and H2B - mCherry captured at early anaphase under different conditions i . e . Control siRNA , knock - down of centralspindlin protein RACGAP1 , in the presence and absence of Aurora B kinase inhibitor ( 2µM ZM ) . Scale bar - 10 µm ( E ) Quantification of aspect ratio of cells treated with different conditions as in ( D ) at 6 mins after anaphase onset , before furrow formation . Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio , while together have a stronger impact during mitotic exit . In combined treatment , mitotic cells hardly undergo any elongation and remain spherical before re - spreading . Ordinary one - way ANOVA with multiple comparison . Each treatment is compared with siControl , * * * * p < 0 . 0001 for all comparisons . Data represented as box - whisker plot , with box showing 25th - 75th percentile values , whiskers - min to max value and line representing median . Mean ± SD for different treatments - siControl ( n = 38 ) - 1 . 455 ± 0 . 19 , ZM ( n = 32 ) - 1 . 219 ± 0 . 16 , siRACGAP1 ( n = 25 ) - 1 . 144 ± 0 . 078 and siRACGAP1 + ZM ( n = 16 ) - 1 . 013 ± 0 . 03 . Cells for different treatment pooled from 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

(D) Still images of representative Hela cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase under different conditions i.e. Control siRNA, knock-down of centralspindlin protein RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). Scale bar - 10 µm (E) Quantification of aspect ratio of cells treated with different conditions as in (D) at 6 mins after anaphase onset, before furrow formation. Aurora B inhibition and siRACGAP1 individually have a moderate effect on aspect ratio, while together have a stronger impact during mitotic exit. In combined treatment, mitotic cells hardly undergo any elongation and remain spherical before re-spreading. Ordinary one-way ANOVA with multiple comparison. Each treatment is compared with siControl, ****p<0.0001 for all comparisons. Data represented as box-whisker plot, with box showing 25th-75th percentile values, whiskers-min to max value and line representing median. Mean±SD for different treatments- siControl (n=38) - 1.455±0.19, ZM (n=32) - 1.219±0.16, siRACGAP1 (n=25) -1.144±0.078 and siRACGAP1+ZM (n=16) - 1.013±0.03. Cells for different treatment pooled from n>3 independent experiments.


( C ) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset , before furrow formation under different conditions as in ( A ) . Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected , while it is cleared upon RACGAP1 depletion . Data represented as mean ± SD for treatments - siControl - 0 . 98 ± 0 . 05 , siRACGAP1 - 0 . 99 ± 0 . 04 , ZM - 1 ± 0 . 039 , siRACGAP1 + ZM - 1 ± 0 . 037 . Ordinary one - way ANOVA with multiple comparison - siControl vs siRACGAP1 - n . s . , p = 0 . 896 , siControl vs ZM - * * p = 0 . 0018 , siControl vs siRACGAP1 + ZM - * * * * p < 0 . 0001 and ZM vs siRACGAP1 + ZM - p = 0 . 1528 .

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

(C) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset, before furrow formation under different conditions as in (A). Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected, while it is cleared upon RACGAP1 depletion. Data represented as mean±SD for treatments - siControl- 0.98±0.05, siRACGAP1 - 0.99±0.04, ZM - 1±0.039, siRACGAP1+ZM - 1±0.037. Ordinary one-way ANOVA with multiple comparison- siControl vs siRACGAP1 - n.s., p=0.896, siControl vs ZM - **p=0.0018, siControl vs siRACGAP1+ZM - ****p<0.0001 and ZM vs siRACGAP1+ZM - p=0.1528.


( G ) Quantification of shortest distance between centroid of DNA and cortical actomyosin network . While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells , the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition . Data represented as mean ± SD . siControl - 4 . 7 ± 0 . 87 , siRACGAP1 - 4 . 4 ± 0 . 64 , ZM - 5 . 47 ± 0 . 87 , siRACGAP1 + ZM - 6 . 35 ± 1 . 14 microns . Ordinary one - way ANOVA with multiple comparison , siControl vs ZM , * * * p = 0 . 002 , siControl vs siRACGAP1 , p = 0 . 2823 , siControl vs siRACGAP1 + ZM , * * * * p < 0 . 0001 , siRACGAP1 vs siRACGAP1 + ZM , * * * * p < 0 . 0001 , ZM vs siRACGAP1 , * * * * p < 0 . 0001 , ZM vs siRACGAP1 + ZM , * * * p = 0 . 0004 .

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 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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

(G) Quantification of shortest distance between centroid of DNA and cortical actomyosin network. While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells, the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition. Data represented as mean±SD. siControl - 4.7±0.87, siRACGAP1 - 4.4±0.64, ZM - 5.47±0.87, siRACGAP1+ZM - 6.35±1.14 microns. Ordinary one-way ANOVA with multiple comparison, siControl vs ZM, ***p=0.002, siControl vs siRACGAP1, p=0.2823, siControl vs siRACGAP1+ZM, ****p<0.0001, siRACGAP1 vs siRACGAP1+ZM, ****p <0.0001, ZM vs siRACGAP1, ****p<0.0001, ZM vs siRACGAP1+ZM, ***p=0.0004 .


( A ) Flow - cytometry for CD1d expression ( top ) and CD1d mean fluorescence intensity ( MFI ; bottom ) on CD45 - cells , ILCs ( Lin - CD45 + CD127 + ) , DCs ( CD11c + ) and B cells ( B220 + ) of the depicted tissues from WT mice ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow-cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45- cells, ILCs (Lin-CD45+CD127+), DCs (CD11c+) and B cells (B220+) of the depicted tissues from WT mice (n=4).


( B ) Flow - cytometry profiles showing gating strategy ( dot - plots ) , CD1d expression ( histograms ) and CD1d MFI ( bars ) in T - bet + ( empty profile ) , GATA - 3 + ( dark grey ) , RORγt + ( light grey ) and T - bet + RORγt + ( dotted line ) ILCs in mLN , spleen , PP and SI - LP ( n = 5 ) . Numbers indicate percentage of cells in the depicted gates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow-cytometry profiles showing gating strategy (dot-plots), CD1d expression (histograms) and CD1d MFI (bars) in T-bet+ (empty profile), GATA-3+ (dark grey), RORγt+ (light grey) and T-bet+RORγt+ (dotted line) ILCs in mLN, spleen, PP and SI-LP (n=5). Numbers indicate percentage of cells in the depicted gates.


( C ) Quantitative RT - PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s ( from mLN of CD1d - deficient mice and mLN and spleen of WT mice ) , B cells and DCs ( n = 3 ) . Results are normalized to those of GAPDH ; RE = relative expression . * * p < 0 . 01 ( two - tailed unpaired t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative RT-PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d-deficient mice and mLN and spleen of WT mice), B cells and DCs (n=3). Results are normalized to those of GAPDH; RE=relative expression. **p< 0.01 (two-tailed unpaired t-test).


( D ) Flow - cytometry profiles showing NKp46 and RORγt expression in Lin - CD45 + cells ; CD1d and CCR6 expression in NCR - ILC3s and CD1d and CD4 expression in NCR - CCR6 + ILC3s from mLN and SI - LP ( n = 4 ) . Numbers indicate percentage of cells in the depicted gates .

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

(D) Flow-cytometry profiles showing NKp46 and RORγt expression in Lin-CD45+cells; CD1d and CCR6 expression in NCR-ILC3s and CD1d and CD4 expression in NCR-CCR6+ILC3s from mLN and SI-LP (n=4). Numbers indicate percentage of cells in the depicted gates.


( E ) CD1d expression ( histograms ) and CD1d MFI in NCR + ( empty profile ) , NCR - CCR6 - ( dark grey ) and NCR - CCR6 + ( light grey ) ILC3s from mLN and SI - LP ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD1d expression (histograms) and CD1d MFI in NCR+ (empty profile), NCR-CCR6- (dark grey) and NCR-CCR6+ (light grey) ILC3s from mLN and SI-LP (n=4).


( A ) Cells were cultured in the presence or absence of BODIPY - αGalCer at 4oC or 37oC as indicated . Flow cytometry profiles and percentages of BODIPY - αGalCer + DC or ILC3 from the indicated tissues incubated at 4oC ( white bars ) or 37oC ( grey bars ) are shown ( n = 3 ) .

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 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

(A) Cells were cultured in the presence or absence of BODIPY-αGalCer at 4oC or 37oC as indicated. Flow cytometry profiles and percentages of BODIPY-αGalCer+ DC or ILC3 from the indicated tissues incubated at 4oC (white bars) or 37oC (grey bars) are shown (n=3).


( B ) WT mice were intravenously injected with BODIPY - αGalCer or PBS ( control ) ; lipid uptake was analysed by flow - cytometry in the depicted populations from the spleen 16 h after lipid injection ( n = 3 ) . Numbers indicate percentage of cells in the depicted gates .

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

(B) WT mice were intravenously injected with BODIPY-αGalCer or PBS (control); lipid uptake was analysed by flow-cytometry in the depicted populations from the spleen 16 h after lipid injection (n=3). Numbers indicate percentage of cells in the depicted gates.


( C ) IL - 2 secretion by DN32 . D3 cells co - cultured with ILC3 sort - purified from mLN or spleen as indicated , pulsed ( + ) or not ( - ) with αGalCer and with or without antiCD1d - blocking 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 B-CONTROLLED_VAR O O O O O O O O O

(C) IL-2 secretion by DN32.D3 cells co-cultured with ILC3 sort-purified from mLN or spleen as indicated, pulsed (+) or not (-) with αGalCer and with or without antiCD1d-blocking antibody.


( D - F ) Sort - purified ILC3s were preincubated with αGalCer ( ILC3 ( αGal ) ) or PBS ( ILC3 ( cont ) ) and adoptively transferred into WT recipients . ( E ) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort - purified from spleen , mLN or SI - LP of mice injected with αGalCer - loaded ILC3s . Gene expression was measured by qPCR , normalized to GAPDH and presented as expression relative to iNKT cells sort - purified from mice injected with control ILC3s ( n = 3 - 5 ) .

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 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

(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (E) Fold change in mRNA expression for the indicated cytokines in iNKT cells sort-purified from spleen, mLN or SI-LP of mice injected with αGalCer-loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort-purified from mice injected with control ILC3s (n=3-5).


( D - F ) Sort - purified ILC3s were preincubated with αGalCer ( ILC3 ( αGal ) ) or PBS ( ILC3 ( cont ) ) and adoptively transferred into WT recipients . ( F ) Intracellular cytokine staining ( left ) and percentage of cytokine + cells ( right ) in spleniciNKT cells from ILC3 - recipient mice ( n = 3 ) . Numbers indicate percentage of cells in the depicted gates . * * p < 0 . 01 , two - tailed unpaired t - test .

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

(D-F) Sort-purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (F) Intracellular cytokine staining (left) and percentage of cytokine+ cells (right) in spleniciNKT cells from ILC3-recipient mice (n=3). Numbers indicate percentage of cells in the depicted gates. **p<0.01, two-tailed unpaired t-test.


( A - B ) Flow - cytometry plots showing gating strategy ( A ) and percentage of ILC populations ( from Lin - CD45 + CD127 + cells , B ) in mLN from WT ( n = 4 ) and CD1d - deficient ( n = 3 ) mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow-cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin-CD45+CD127+cells, B) in mLN from WT (n=4) and CD1d-deficient (n=3) mice.


( C ) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody - mediated CD1d crosslink ( n = 3 ) . Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s . * p < 0 . 05 two - tailed unpaired t - test .

(C) Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody-mediated CD1d crosslink (n=3). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. *p<0.05 two-tailed unpaired t-test.


( D ) Fold change in IL22 mRNA expression in ILC3s after antibody - mediated CD1d crosslink and / or IL - 23 stimulation ( n = 4 ) . Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s . * * p < 0 . 01 two - tailed unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fold change in IL22 mRNA expression in ILC3s after antibody-mediated CD1d crosslink and/or IL-23 stimulation (n=4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. **p<0.01 two-tailed unpaired t-test.


( E ) IL - 22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and / or IL - 23 ( n = 3 ) . * p < 0 . 05 two - tailed unpaired 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

(E) IL-22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and/or IL-23 (n=3). *p<0.05 two-tailed unpaired t-test


( F - G ) Intracellular cytokine staining ( F ) and percentage of IL - 22 + ILC3s ( G ) after stimulation with αCD1d , IL - 23 and / or IL - 1β . * p < 0 . 05 , * * p < 0 . 01 two - tailed unpaired t - test ( n = 4 - 8 )

(F-G) Intracellular cytokine staining (F) and percentage of IL-22+ILC3s (G) after stimulation with αCD1d, IL-23 and/or IL-1β. *p<0.05, **p<0.01 two-tailed unpaired t-test (n=4-8)


( H ) WT mice were i . v . injected with αGalCer or PBS ( control ) and activation of splenicILC3s was assessed by flow - cytometry as CD69 up - regulation at 6 h or 16 h after injection . Grey filled profile = ILC3 from αGalCer - injected mice ; Empty profile = ILC3 from PBS - injected mice ; Dotted line = CD45 - cells . Right graph , percentage of CD69 + ILC3s 6 h after injection of αGalCer ( grey ) or PBS ( white ) . ( n = 3 ) * p < 0 . 05 two - tailed unpaired t - test .

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 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 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

(H) WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenicILC3s was assessed by flow-cytometry as CD69 up-regulation at 6 h or 16 h after injection. Grey filled profile= ILC3 from αGalCer-injected mice; Empty profile= ILC3 from PBS-injected mice; Dotted line= CD45-cells. Right graph, percentage of CD69+ILC3s 6 h after injection of αGalCer (grey) or PBS (white). (n=3) *p<0.05 two-tailed unpaired t-test.


( I - J ) Fold change in mRNA expression for the indicated cytokines in ILC3s sort - purified from spleen ( I ) or SI - LP ( J ) 6 h after intravenous ( I ) or oral ( J ) αGalcer administration . Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort - purified from PBS - injected mice ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 two - tailed unpaired t - test .

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

(I-J) Fold change in mRNA expression for the indicated cytokines in ILC3s sort-purified from spleen (I) or SI-LP (J) 6 h after intravenous (I) or oral (J) αGalcer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort-purified from PBS-injected mice (n=3). *p<0.05, **p<0.01 two-tailed unpaired t-test.


B , C Serial transplantations of PR + / + and PR − / − mammary epithelia . ( B ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from 8 ‐ week ‐ old PR + / + ; EGFP and PR − / − ; EGFP donor mice . ( C ) Table summarizing 3 independent serial transplant experiments with PR + / + ; EGFP and PR − / − ; EGFP . Each engrafted gland is represented by a micrograph ; black sectors represent area of fat pad filled by engrafted epithelium . Scale bar : 200 μm .

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

B, C Serial transplantations of PR+/+ and PR−/− mammary epithelia. (B) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 8‐week‐old PR+/+; EGFP and PR−/−; EGFP donor mice. (C) Table summarizing 3 independent serial transplant experiments with PR+/+; EGFP and PR−/−; EGFP. Each engrafted gland is represented by a micrograph; black sectors represent area of fat pad filled by engrafted epithelium. Scale bar: 200 μm.


D , E Serial transplantation of RANKL + / + and RANKL − / − mammary epithelia . ( D ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from 5 ‐ week ‐ old RANKL + / + ; EGFP and RANKL − / − ; EGFP donor mice . Insets : higher magnification showing side branches present in the WT control ( arrowheads ) absent from RANKL − / − ; EGFP epithelium . ( E ) Table summarizing three independent serial transplant experiments with RANKL + / + ; EGFP and RANKL − / − ; EGFP donor mice . Scale bar : 200 μm .

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

D, E Serial transplantation of RANKL+/+ and RANKL−/− mammary epithelia. (D) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from 5‐week‐old RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Insets: higher magnification showing side branches present in the WT control (arrowheads) absent from RANKL−/−; EGFP epithelium. (E) Table summarizing three independent serial transplant experiments with RANKL+/+; EGFP and RANKL−/−; EGFP donor mice. Scale bar: 200 μm.


F , G Serial transplantation of Wnt4 + / + and Wnt4 − / − mammary epithelia . ( F ) Fluorescence stereo microscopy of third ‐ generation mammary outgrowths derived from mammary buds of E12 . 5 and E13 . 5 Wnt4 + / + ; EGFP and Wnt4 − / − ; EGFP embryos . Scale bar : 200 μm . ( G ) Table summarizing three independent serial transplant experiments with Wnt4 + / + ; EGFP donor mice . Scale bar : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G Serial transplantation of Wnt4+/+ and Wnt4−/− mammary epithelia. (F) Fluorescence stereo microscopy of third‐generation mammary outgrowths derived from mammary buds of E12.5 and E13.5 Wnt4+/+; EGFP and Wnt4−/−; EGFP embryos. Scale bar: 200 μm. (G) Table summarizing three independent serial transplant experiments with Wnt4+/+; EGFP donor mice. Scale bar: 200 μm.


H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation . P values were determined by Mann - Whitney U ‐ test .

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

H Box plot showing the difference between percentage of reconstitution between WT and MT contralateral grafts in each transplant generation. P values were determined by Mann-Whitney U‐test.


A Epifluorescence stereo microscopy of inguinal mammary gland from a 5 ‐ day ‐ old Wnt4 : : Cre ; mT / mG female ( n = 7 ) . Scale bars : 0 . 5 mm and 0 . 1 mm ( inset ) .

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

A Epifluorescence stereo microscopy of inguinal mammary gland from a 5‐day‐old Wnt4::Cre; mT/mG female (n = 7). Scale bars: 0.5 mm and 0.1 mm (inset).


B , C Histology sections of mammary glands from a 5 ‐ day ‐ old ( B ; n = 7 ) and a 10 ‐ day ‐ old ( C ; n = 5 ) Wnt4 : : Cre ; mT / mG female stained by double immunofluorescence microscopy for EGFP ( green ) and PR ( magenta , not detected ) , counterstained with DAPI ( blue ) . Scale bar : 50 mm .

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

B, C Histology sections of mammary glands from a 5‐day‐old (B; n = 7) and a 10‐day‐old (C; n = 5) Wnt4::Cre; mT/mG female stained by double immunofluorescence microscopy for EGFP (green) and PR (magenta, not detected), counterstained with DAPI (blue). Scale bar: 50 mm.


D - F EGFP ( green ) and PR ( magenta ) co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histology sections from mT / mG ; Wnt4 : : Cre mammary glands at different developmental stages . ( D ) TEB of a 4 ‐ week ‐ old female ( n = 4 ) ; scale bar : 30 μm . ( E ) Ducts of an 8 ‐ week ‐ old female ( n = 3 ) ; scale bar : 100 μm ; inset , scale bar : 20 μm . ( F ) Duct of a female at day 10 . 5 of pregnancy ( n = 3 ) ; scale bar : 150 μm ; inset , scale bar : 30 μm .

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

D-F EGFP (green) and PR (magenta) co‐immunofluorescence microscopy counterstained with DAPI (blue) on histology sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (D) TEB of a 4‐week‐old female (n = 4); scale bar: 30 μm. (E) Ducts of an 8‐week‐old female (n = 3); scale bar: 100 μm; inset, scale bar: 20 μm. (F) Duct of a female at day 10.5 of pregnancy (n = 3); scale bar: 150 μm; inset, scale bar: 30 μm.


G - I EGFP ( green ) , PR ( magenta ) , and p63 ( white ) triple co ‐ immunofluorescence microscopy counterstained with DAPI ( blue ) on histological sections from mT / mG ; Wnt4 : : Cre mammary glands at different developmental stages . ( G ) Ducts of 5 ‐ day ‐ old female ( n = 3 ) . ( H ) TEB of a 4 ‐ week ‐ old female ( n = 3 ) . ( I ) Duct of an 8 ‐ week ‐ old female ( n = 3 ) . Scale bars : 30 μm .

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

G-I EGFP (green), PR (magenta), and p63 (white) triple co‐immunofluorescence microscopy counterstained with DAPI (blue) on histological sections from mT/mG; Wnt4::Cre mammary glands at different developmental stages. (G) Ducts of 5‐day‐old female (n = 3). (H) TEB of a 4‐week‐old female (n = 3). (I) Duct of an 8‐week‐old female (n = 3). Scale bars: 30 μm.


J - O Epifluorescence stereo microscopy of mammary glands harvested from 15 ‐ day ‐ old Wnt4 : : GFP females either WT ( n = 18 ) ( J , M ) , ERα − / − ( n = 4 ) ( K , N ) , or PR − / − ( n = 3 ) ( L , O ) . dTomato expression ( J - L ) ; EGFP expression ( M - O ) is not abrogated in ERα − / − nor PR − / − epithelia . Arrowheads mark the main duct originating from the nipple . Scale bar : 50 μm .

J-O Epifluorescence stereo microscopy of mammary glands harvested from 15‐day‐old Wnt4::GFP females either WT (n = 18) (J, M), ERα−/− (n = 4) (K, N), or PR−/− (n = 3) (L, O). dTomato expression (J-L); EGFP expression (M-O) is not abrogated in ERα−/− nor PR−/− epithelia. Arrowheads mark the main duct originating from the nipple. Scale bar: 50 μm.


Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal ( 6 weeks old ) and 3 adult ( 11 weeks old ) mice exposed for 6 h to vehicle ( C ) , 17β ‐ estradiol ( 20 nmol ) ( E2 ) , or R5020 ( 20 nmol ) ( P ) . Bars represent the mean ± SD of 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Q Bar plots showing relative PR and Wnt4 mRNA expression normalized to CK18 mRNA in mammary organoids from 5 pubertal (6 weeks old) and 3 adult (11 weeks old) mice exposed for 6 h to vehicle (C), 17β‐estradiol (20 nmol) (E2), or R5020 (20 nmol) (P). Bars represent the mean ± SD of 3 independent experiments.


R - X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4 : : GFP epithelium from 8 ‐ week ‐ old females , either PRWT ( R , T , V , X ) or PR − / − ( S , U , W ) . dTomato expression ( R , S ) ; EGFP expression ( T , U ) double epifluorescence ( V , W , X ) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old . Representative result from three independent experiments . Arrowheads point to TEBs ( V , X ) or to origin of growth ( W ) . Scale bar ( R - W ) : 5 mm , ( X ) : 1 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-X Epifluorescence stereo microscopy of contralateral mammary glands that were engrafted with Wnt4::GFP epithelium from 8‐week‐old females, either PRWT (R, T, V, X) or PR−/− (S, U, W). dTomato expression (R, S); EGFP expression (T, U) double epifluorescence (V, W, X) on contralateral engrafted glands 3 weeks after surgery when recipients were 6 weeks old. Representative result from three independent experiments. Arrowheads point to TEBs (V, X) or to origin of growth (W). Scale bar (R-W): 5 mm, (X): 1 mm.


A - D Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10 ‐ day ‐ old mT / mG ( A , C ) and MMTV : : Cre ; mT / mG ( B , D ) female ( n = 7 ) . Scale bar : 1 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative epifluorescence stereo microscopy of inguinal mammary glands from a 10‐day‐old mT/mG (A, C) and MMTV::Cre; mT/mG (B, D) female (n = 7). Scale bar: 1 mm.


E Higher magnification of inguinal mammary gland from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG female . Scale bar : 0 . 2 mm . F Immunofluorescence microscopy for EGFP ( green ) on a mammary gland section from a 10 ‐ day ‐ old MMTV : : Cre ; mT / mG female counterstained with DAPI ( blue ) ( n = 7 ) . Scale bar : 30 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

E Higher magnification of inguinal mammary gland from a 10‐day‐old MMTV::Cre; mT/mG female. Scale bar: 0.2 mm.F Immunofluorescence microscopy for EGFP (green) on a mammary gland section from a 10‐day‐old MMTV::Cre; mT/mG female counterstained with DAPI (blue) (n = 7). Scale bar: 30 μm.


G Epifluorescence stereo microscopy of mammary glands from 10 ‐ day ‐ old littermates either MMTV : : Cre ; mT / mG Wnt4fl / + or MMTV : : Cre ; mT / mG ; Wnt4fl / fl . Scale bar : 1 mm . Arrowhead marks the main duct originating from the nipple . H Bar plot showing ratio of branching points in prepubertal Cre + ; Wnt4fl / fl ( n = 12 ) relative to Cre + ; Wnt4fl / + littermates ( n = 10 ) . Two ‐ tailed , paired Student ' s t ‐ test was used to calculate statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Epifluorescence stereo microscopy of mammary glands from 10‐day‐old littermates either MMTV::Cre; mT/mG Wnt4fl/+ or MMTV::Cre; mT/mG; Wnt4fl/fl. Scale bar: 1 mm. Arrowhead marks the main duct originating from the nipple.H Bar plot showing ratio of branching points in prepubertal Cre+;Wnt4fl/fl (n = 12) relative to Cre+;Wnt4fl/+ littermates (n = 10). Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


I Fluorescence stereo microscopy of mammary glands from 35 ‐ day ‐ old littermates either MMTV : : Cre ; mT / m ; Wnt4fl / + or MMTV : : Cre ; mT / m ; Wnt4fl / fl . Note ductal elongation is delayed in Wnt4 ‐ deficient mammary epithelium compared to Wnt4fl / + control . LN : lymph node . Scale bar : 4 mm . J , K Bar plots showing number of terminal end buds ( TEBs ) ( J ) and area of mammary fat pad filled by the ductal system ( K ) . Ctrl ( Wnt4fl / wt or Wnt4fl / fl ; Cre − ) : n = 6 , fl / + ; Cre + : n = 4 , and fl / fl ; Cre + : n = 7 ) . Data are presented as the mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to calculate statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence stereo microscopy of mammary glands from 35‐day‐old littermates either MMTV::Cre; mT/m; Wnt4fl/+ or MMTV::Cre; mT/m;Wnt4fl/fl. Note ductal elongation is delayed in Wnt4‐deficient mammary epithelium compared to Wnt4fl/+ control. LN: lymph node. Scale bar: 4 mm.J, K Bar plots showing number of terminal end buds (TEBs) (J) and area of mammary fat pad filled by the ductal system (K). Ctrl (Wnt4fl/wt or Wnt4fl/fl; Cre−): n = 6, fl/+; Cre+: n = 4, and fl/fl; Cre+: n = 7). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


L , M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4 ‐ week ‐ old Wnt4fl / + ; Cre + ( n = 3 ) or Wnt4fl / fl ; Cre + ( n = 4 ) in the ducts ( L ) and in the TEBs ( M ) . Data are presented as the mean ± SD . Two ‐ tailed Student ' s t ‐ test was used to calculate statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, M Bar plots showing BrdU incorporation index in inguinal mammary glands of 4‐week‐old Wnt4fl/+; Cre+ (n = 3) or Wnt4fl/fl; Cre+ (n = 4) in the ducts (L) and in the TEBs (M). Data are presented as the mean ± SD. Two‐tailed Student's t‐test was used to calculate statistical significance.


A Whole ‐ mount microscopy of X ‐ gal ‐ stained Axin2 + / lacZ in E12 . 5 embryo showing β ‐ galactosidase expression in the mammary buds ( arrows ) ( n = 8 ) . Scale bar : 1 mm . Arrowheads mark mammary buds .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Whole‐mount microscopy of X‐gal‐stained Axin2+/lacZ in E12.5 embryo showing β‐galactosidase expression in the mammary buds (arrows) (n = 8). Scale bar: 1 mm. Arrowheads mark mammary buds.


B - F Whole ‐ mount microscopy of X ‐ gal ( blue ) ‐ and carmine alum ( red ) ‐ stained mammary glands harvested from Axin2 + / lacZ mice at distinct developmental stages . ( B ) At postnatal day 1 , β ‐ galactosidase activity detected in the nipple area ( n = 6 ) . Scale bar : 1 mm . ( C , D ) In 5 ‐ week ‐ old mammary glands , reporter activity was detected around the ducts ( small arrows ) and in the neck region of the terminal end buds ( TEBs ) ( large arrows ) ( C ) ( n = 8 ) . Scale bars : 400 μm ( C ) and 100 μm ( D ) . ( E ) At 8 . 5 day of pregnancy , reporter expression was detected in the ducts . Higher magnification ( inset ) suggests myoepithelial expression ( n = 10 ) . Scale bar : 1 mm . ( F ) Whole ‐ mount at day 14 . 5 of pregnancy : reporter activity is limited to ducts ( n = 5 ) . Scale bar : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Whole‐mount microscopy of X‐gal (blue)‐ and carmine alum (red)‐stained mammary glands harvested from Axin2+/lacZ mice at distinct developmental stages. (B) At postnatal day 1, β‐galactosidase activity detected in the nipple area (n = 6). Scale bar: 1 mm. (C, D) In 5‐week‐old mammary glands, reporter activity was detected around the ducts (small arrows) and in the neck region of the terminal end buds (TEBs) (large arrows) (C) (n = 8). Scale bars: 400 μm (C) and 100 μm (D). (E) At 8.5 day of pregnancy, reporter expression was detected in the ducts. Higher magnification (inset) suggests myoepithelial expression (n = 10). Scale bar: 1 mm. (F) Whole‐mount at day 14.5 of pregnancy: reporter activity is limited to ducts (n = 5). Scale bar: 200 μm.


G Histology section of Axin2 : : LacZ mammary gland at day 8 . 5 of pregnancy counterstained with nuclear red ; luminal epithelial cells show no detectable β ‐ galactosidase activity but myoepithelial cells do . Scale bar : 200 μm .

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

G Histology section of Axin2::LacZ mammary gland at day 8.5 of pregnancy counterstained with nuclear red; luminal epithelial cells show no detectable β‐galactosidase activity but myoepithelial cells do. Scale bar: 200 μm.


H β ‐ galactosidase activity ( blue ) colocalizes with the myoepithelial marker p63 ( green ) detected by immunofluorescence microscopy . Arrows point to myoepithelial cells . L , lumen . Scale bar : 100 μm .

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

H β‐galactosidase activity (blue) colocalizes with the myoepithelial marker p63 (green) detected by immunofluorescence microscopy. Arrows point to myoepithelial cells. L, lumen. Scale bar: 100 μm.


I Representative whole ‐ mount stereo microscopy of X ‐ gal ( blue ) ‐ and carmine alum ( magenta ) ‐ stained mammary gland biopsies taken from 14 ‐ week ‐ old Axin2 : : LacZ females collected at diestrus and estrus , respectively ( n = 3 ) . Scale bar : 200 μm .

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

I Representative whole‐mount stereo microscopy of X‐gal (blue)‐ and carmine alum (magenta)‐stained mammary gland biopsies taken from 14‐week‐old Axin2::LacZ females collected at diestrus and estrus, respectively (n = 3). Scale bar: 200 μm.


J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT - PCR normalized to 18S rRNA . Two ‐ tailed , paired Student ' s t ‐ test was used to calculate statistical significance .

J Relative Wnt4 and Axin2 mRNA expression in mammary glands from three mice in estrus versus diestrus assessed by semiquantitative qRT-PCR normalized to 18S rRNA. Two‐tailed, paired Student's t‐test was used to calculate statistical significance.


K Stereo microscopy of X ‐ gal ‐ and carmine alum ‐ stained mammary glands from ovariectomized Axin2 : : LacZ females treated for 72 h with vehicle ( n = 4 ) ( left ) , 17 ‐ β ‐ estradiol ( E2 ) ( n = 6 ) ( center ) , 17 ‐ β ‐ estradiol and progesterone ( E2 and P ) ( n = 8 ) ( right ) . Scale bar : 200 μm .

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

K Stereo microscopy of X‐gal‐ and carmine alum‐stained mammary glands from ovariectomized Axin2::LacZ females treated for 72 h with vehicle (n = 4) (left), 17‐β‐estradiol (E2) (n = 6) (center), 17‐β‐estradiol and progesterone (E2 and P) (n = 8) (right). Scale bar: 200 μm.


L , M Stereo microscopy of contralateral glands whole ‐ mounted and X ‐ gal stained after engraftment with mammary buds from Axin2 : : LacZ transgenic and Wnt4 + / + or Wnt4 − / − female E12 . 5 and E13 . 5 embryos ( L ) or Axin2 : : LacZ transgenic and either PR + / − or PR − / − 8 ‐ week ‐ old females ( M ) , at day 8 . 5 of pregnancy . β ‐ galactosidase expression reflecting Axin2 transcription is readily detected in PR + / − as well as Wnt4 + / + mammary epithelia but not in the PR − / − ( n = 6 ) and Wnt4 − / − counterparts ( n = 6 ) . Blue : X ‐ gal staining ; magenta : carmine alum counterstain . Scale bars : 200 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, M Stereo microscopy of contralateral glands whole‐mounted and X‐gal stained after engraftment with mammary buds from Axin2::LacZ transgenic and Wnt4+/+ or Wnt4−/− female E12.5 and E13.5 embryos (L) or Axin2::LacZ transgenic and either PR+/− or PR−/− 8‐week‐old females (M), at day 8.5 of pregnancy. β‐galactosidase expression reflecting Axin2 transcription is readily detected in PR+/− as well as Wnt4+/+ mammary epithelia but not in the PR−/− (n = 6) and Wnt4−/− counterparts (n = 6). Blue: X‐gal staining; magenta: carmine alum counterstain. Scale bars: 200 μm.


A X ‐ gal ‐ stained whole ‐ mounts of Axin2 : : LacZ embryos at E14 . 5 either Wnt4 + / + or Wnt4 − / − ( n = 5 or 4 , respectively ) . Arrows mark mammary buds . Scale bar : 5 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 X‐gal‐stained whole‐mounts of Axin2::LacZ embryos at E14.5 either Wnt4+/+or Wnt4−/− (n = 5 or 4, respectively). Arrows mark mammary buds. Scale bar: 5 mm.