Figure 3 ( A - C ) Embryos co - expressing the optogenetic module and Sqh : : mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo - activation of the cell base in the anterior ( left ) half of the embryo and confocal recording of the whole embryo . The experiment was started at the end of cellularization when initial accumulation of apical myosin - II was detected and terminated at different time points to record the plasma membrane signal ( CIBN : : GFP - pm ) in the whole embryo using two - photon illumination . Still frames visualizing the plasma membrane ( CIBN : : GFP - pm ) at a sub - apical plane at the initial time point of the experiment ( A ) , 3 min ( B ) and 10 min ( C ) after initial photo - activation .

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

Figure 3(A-C) Embryos co-expressing the optogenetic module and Sqh::mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo-activation of the cell base in the anterior (left) half of the embryo and confocal recording of the whole embryo. The experiment was started at the end of cellularization when initial accumulation of apical myosin-II was detected and terminated at different time points to record the plasma membrane signal (CIBN::GFP-pm) in the whole embryo using two-photon illumination. Still frames visualizing the plasma membrane (CIBN::GFP-pm) at a sub-apical plane at the initial time point of the experiment (A), 3 min (B) and 10 min (C) after initial photo-activation.


Figure 4Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( A ) Still frames of the time lapse membrane ( GAP43 : : mCherry ) recording are shown at the initial time point ( top ) , after 6 . 5 min ( middle ) , and after 13 min ( bottom ) of photo - activation . Cells in the non - activated region ( green ) underwent normal apical constriction . In the activated region , two populations of cells were observed , one that constricted ( purple ) and one that did not constrict ( red ) . Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells . Cells that constricted less were considered to be defective in apical constriction and are referred to as non - constricting . Scale bar 20 µm . Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( B ) All cells show pulsatile behavior . Graphs illustrate the area of three representative cells in each category ( non - activated region : top ; activated region with constricting cells : middle ; activated region with non - constricting cells : bottom ) over the course of one experiment . Yellow circles indicate the position of automatically identified local maxima used for further analyses . Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( C , D ) ( C ) Diagram showing the extent of ratchet - like constrictions . The mean difference in apical area between adjacent peaks ( relaxed cell surface ) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse . Cells in the non - activated region ( NR ) constricted on average more than 5 µm2 , which is characteristic for ratchet - like constriction . Non - constricting cells in the activated region ( AR , non - con . ) displayed a mean difference in apical area per pulse of about 0 , reflecting non - ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size . Constricting cells in the activated region ( AR , con . ) show an intermediate behavior with less efficient constrictions than in the NR . Data points are significantly different as determined by ANOVA ( F ( 2 , 539 ) = 72 . 04 , p = 1 . 9e - 28 ) and post - hoc Tukey test . ( D ) The pulsation period ( T ) . All cells pulse with a period of about 90 s . There were no statistically significant differences between group means as determined by one - way ANOVA ( F ( 2 , 540 ) = 1 . 438 , p = 0 . 24 ) . ( C and D ) Red dots indicate the mean values and the error bars represent the standard deviation . More than 500 pulses ( nNR = 119 , nAR , non - con . = 128 , nAR , con = 295 ) of more than 100 cells were analyzed . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test . Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( E ) Diagram showing the prevalence of cells in the photo - activated region that constricted ( con . ) and that did not constrict ( Non - con . ) . More than 4 / 5 of the cells did not constrict to half of the initial area ( N = 5 embryos ; n : 407 cells ; Cohen ' s D : d = 10 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test . Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( F , G ) Graphs showing the average apical area ( F ) and anisotropy ( G ) of cells in the non - activated region ( green ) , in the activated region that constricted ( purple ) and that did not constrict ( red ) . The straight line indicates the mean value at a given time - point , the semi - transparent colored area indicated the respective standard deviation . Cells in the non - activated region constricted efficiently and progressively acquired anisotropy . Constricting cells in the activated region constricted slower in an isotropic manner . Non - constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy ( nNR = 34 , nAR , non - con . = 20 , nAR , con = 59 ) .

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

Figure 4Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (A) Still frames of the time lapse membrane (GAP43::mCherry) recording are shown at the initial time point (top), after 6.5 min (middle), and after 13 min (bottom) of photo-activation. Cells in the non-activated region (green) underwent normal apical constriction. In the activated region, two populations of cells were observed, one that constricted (purple) and one that did not constrict (red). Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells. Cells that constricted less were considered to be defective in apical constriction and are referred to as non-constricting. Scale bar 20 µm. Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (B) All cells show pulsatile behavior. Graphs illustrate the area of three representative cells in each category (non-activated region: top; activated region with constricting cells: middle; activated region with non-constricting cells: bottom) over the course of one experiment. Yellow circles indicate the position of automatically identified local maxima used for further analyses.Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (C, D) (C) Diagram showing the extent of ratchet-like constrictions. The mean difference in apical area between adjacent peaks (relaxed cell surface) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse. Cells in the non-activated region (NR) constricted on average more than 5 µm2, which is characteristic for ratchet-like constriction. Non-constricting cells in the activated region (AR, non-con.) displayed a mean difference in apical area per pulse of about 0, reflecting non-ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size. Constricting cells in the activated region (AR, con.) show an intermediate behavior with less efficient constrictions than in the NR. Data points are significantly different as determined by ANOVA (F(2,539)=72.04, p=1.9e-28) and post-hoc Tukey test. (D) The pulsation period (T). All cells pulse with a period of about 90 s. There were no statistically significant differences between group means as determined by one-way ANOVA (F(2,540)=1.438, p=0.24). (C and D) Red dots indicate the mean values and the error bars represent the standard deviation. More than 500 pulses (nNR = 119, nAR, non-con. = 128, nAR, con = 295) of more than 100 cells were analyzed. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (E) Diagram showing the prevalence of cells in the photo-activated region that constricted (con.) and that did not constrict (Non-con.). More than 4/5 of the cells did not constrict to half of the initial area (N = 5 embryos; n: 407 cells; Cohen's D: d = 10). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (F,G) Graphs showing the average apical area (F) and anisotropy (G) of cells in the non-activated region (green), in the activated region that constricted (purple) and that did not constrict (red). The straight line indicates the mean value at a given time-point, the semi-transparent colored area indicated the respective standard deviation. Cells in the non-activated region constricted efficiently and progressively acquired anisotropy. Constricting cells in the activated region constricted slower in an isotropic manner. Non-constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy (nNR = 34, nAR, non-con. = 20, nAR, con = 59).


Figure 5 ( C - E ) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2 - CRY2 : : mCherry were imaged during late cellularization . ( C ) z - projections of confocal image stacks show the localization of the respective GFP - tagged optogenetic anchor . Please note that the construct PatJ - CIBN ( indicated by asterisk ) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2 - CRY2 : : mCh . All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation . Scale bars , 20 μm . ( D ) A region of interest ( right side of the dashed line ) in a 10 µm - sized stack was photo - activated using two - photon illumination for 30 s before acquiring the RhoGEF2 - CRY2 : : mCherry signal using confocal microscopy . All anchor proteins were able to recruit RhoGEF2 - CRY2 : : mCherry specifically to the activated region . Scale bars , 10 μm . ( E ) Although all basal anchors were effective at recruiting RhoGEF2 - CRY2 : : mCherry to the cell base , only PatJ - CIBN : : GFP - pm resulted in effective myosin - II recruitment and actomyosin ring constriction

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

Figure 5 (C-E) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2-CRY2::mCherry were imaged during late cellularization. (C) z-projections of confocal image stacks show the localization of the respective GFP-tagged optogenetic anchor. Please note that the construct PatJ-CIBN (indicated by asterisk) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2-CRY2::mCh. All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation. Scale bars, 20 μm. (D) A region of interest (right side of the dashed line) in a 10 µm-sized stack was photo-activated using two-photon illumination for 30 s before acquiring the RhoGEF2-CRY2::mCherry signal using confocal microscopy. All anchor proteins were able to recruit RhoGEF2-CRY2::mCherry specifically to the activated region. Scale bars, 10 μm. (E) Although all basal anchors were effective at recruiting RhoGEF2-CRY2::mCherry to the cell base, only PatJ-CIBN::GFP-pm resulted in effective myosin-II recruitment and actomyosin ring constriction


Figure 6 ( A ) Localization of the basal - specific optogenetic anchor CIBN - PatJ : : GFP - pm during ventral furrow formation . Embryos expressing CIBN - PatJ : : GFP - pm were imaged using two - photon excitation during ventral furrow formation . Transverse cross section ( z - projection ) at a stage when cells were already apically constricted and elongated , and the ventral tissue already internalized . Embryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY2 and Sqh : : mCherry ( B , C were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation . ( B ) myosin - II distribution in a cross section ( z - projection ) 3 min after photo - activation . myosin - II was locally recruited to the base as indicated by the red arrowheads in the activated region ( red line ) . ( C ) Top view ( sum of slice of 4 µm - sized image stack ) showing basal myosin - II recruitment in the photo - activated region ( red dashed square ) Data information : Scale bars , 20 µmEmbryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY an the membrane marker GAP43 : : mCherry ( D - I ) were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation ( D ) Cross section ( z - projection ) showing the plasma membrane and ( E ) top view showing the apical plasma membrane at the initial time point before photo - activation . At this stage , the central mesodermal tissue has started to internalize and cells were constricted at the apical surface . In an alternating fashion , the base of the cells in the anterior half of the embryo ( left ) was photo - activated for 100 sec and the GAP43 : : mCherry signal in the entire field of view was recorded . The furrow further ingressed and closed completely 3 min ( F ) and 6 min ( G ) after initial activation , respectively . In the activated region , the tissue failed to internalize and to form a closed furrow both at 3 min ( H ) and 6 min ( I ) after initial photo - activation . The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm ( D ) . In the non - activated region 6 min after initial activation the width of the tissue shrank to about 25 µm ( G ) , whereas it remained constant at 40 µm in the activated region ( I ) . Data information : Scale bars , 20

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

Figure 6(A) Localization of the basal-specific optogenetic anchor CIBN-PatJ::GFP-pm during ventral furrow formation. Embryos expressing CIBN-PatJ::GFP-pm were imaged using two-photon excitation during ventral furrow formation. Transverse cross section (z-projection) at a stage when cells were already apically constricted and elongated, and the ventral tissue already internalized.Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY2 and Sqh::mCherry (B,C were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation. (B) myosin-II distribution in a cross section (z-projection) 3 min after photo-activation. myosin-II was locally recruited to the base as indicated by the red arrowheads in the activated region (red line). (C) Top view (sum of slice of 4 µm-sized image stack) showing basal myosin-II recruitment in the photo-activated region (red dashed square) Data information: Scale bars, 20 µmEmbryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY an the membrane marker GAP43::mCherry (D-I) were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation (D) Cross section (z-projection) showing the plasma membrane and (E) top view showing the apical plasma membrane at the initial time point before photo-activation. At this stage, the central mesodermal tissue has started to internalize and cells were constricted at the apical surface. In an alternating fashion, the base of the cells in the anterior half of the embryo (left) was photo-activated for 100 sec and the GAP43::mCherry signal in the entire field of view was recorded. The furrow further ingressed and closed completely 3 min (F) and 6 min (G) after initial activation, respectively. In the activated region, the tissue failed to internalize and to form a closed furrow both at 3 min (H) and 6 min (I) after initial photo-activation. The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm (D). In the non-activated region 6 min after initial activation the width of the tissue shrank to about 25 µm (G), whereas it remained constant at 40 µm in the activated region (I). Data information: Scale bars, 20 µm


Figure 1A , B Quantification of mitotic ( A ) and meiotic ( B ) cells in the distal germline in wild - type animals fed bacteria transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) Data information : Data are presented as mean ± SE ( A ) N = 3 at least 5 worms per replicate and condition , ( B ) N = 3 at least 10 worms for conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsQuantification of dissected distal germline from wild - type animal stained either anti - phosphohistone H3 ( anti - PH3 ) ( C relative to the total number of mitotic cells of wild - type animal Data informatio Data are presented a as mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsQuantification of dissected distal germline from wild - type animal stained eithe anti - CDK - 1 ( anti - CDK - 1 ( D relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsQuantification of dissected distal germline from wild - type animal stained eithe 5 - ethynyl - 2 ′ - deoxyuridine ( EdU - green ) ( E ) relative to the total number of mitotic cells of wild - type animal Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate and conditio * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple comparisonsF Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild - type worms fed as in ( A ) Data information : Data are presented as mean ± SE ( F ) N = 5 at least 10 worms per replicate and condition . * P < 0 . 05 and * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

Figure 1A, B Quantification of mitotic (A) and meiotic (B) cells in the distal germline in wild-type animals fed bacteria transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) Data information: Data are presented as mean ± SE (A) N=3 at least 5 worms per replicate and condition, (B) N=3 at least 10 worms for conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsQuantification of dissected distal germline from wild-type animal stained either anti-phosphohistone H3 (anti-PH3) (C relative to the total number of mitotic cells of wild-type animal Data informatio Data are presented a as mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsQuantification of dissected distal germline from wild-type animal stained eithe anti-CDK-1 (anti-CDK-1 (D relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsQuantification of dissected distal germline from wild-type animal stained eithe 5-ethynyl-2′-deoxyuridine (EdU -green) (E) relative to the total number of mitotic cells of wild-type animal Data information: Data are presented a mean ± S N=2 at least 5 worms per replicate and conditio * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisonsF Average number of apoptotic corpses in the meiotic compartment relative to the total number of meiotic cells in wild-type worms fed as in (A) Data information: Data are presented as mean ± SE (F) N=5 at least 10 worms per replicate and condition. * P < 0.05 and **** P < 0.0001 vs con (One-way ANOVA Tukey"s multiple comparisons


Figure 2Quantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiation 125 Gy ( A Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit ) , UVC 400J / m2 ( B Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment o wild - type strain relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit different doses of UVB ( C Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED - 1 : : GF relative to the total number of meiotic cells , fed bacteria transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit 100µM cisplatin Data information : Data are presented as mean ± SEM . For each panel at least N = 3 and 10 worms for replicate and condition . * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Figure 2Quantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiation 125 Gy (A Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit ), UVC 400J/m2 (B Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment o wild-type strain relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit different doses of UVB (C Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsQuantification of apoptotic corpse in the meiotic compartment of CED-1::GF relative to the total number of meiotic cells, fed bacteria transformed with either empty-vector (con) or vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit 100µM cisplatin Data information: Data are presented as mean ± SEM. For each panel at least N=3 and 10 worms for replicate and condition. * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ## <0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisons


Figure 3Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 ( A and left untreated or exposed to 1200 J / m2 UVB . Apoptotic cells were counted 12 , 24 and 36 hours after exposure Data information : Data are presented as mean ± SEM , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsTime course quantification of apoptotic corpses relative to the total number of meitic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) o isp - 1 ( B ) , and left untreated or exposed to 1200 J / m2 UVB . Apoptotic cells were counted 12 , 24 and 36 hours after exposure Data information : Data are presented as mean ± SEM , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOV Tukey " s multiple comparisonsReal Time PCR gene expression analysis of egl - genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiatio Data information : Data are presented a mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsReal Time PCR gene expression analysis of egl - genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated wit UV Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsReal Time PCR gene expression analysis o ced - 1 genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treated with gamma radiatio Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonsReal Time PCR gene expression analysis o ced - 1 genes , performed in wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 and left untreated or treate with UV Data information : Data are presented as mean ± SE N = F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 and # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s mu comparisons

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

Figure 3Time course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 (A and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsTime course quantification of apoptotic corpses relative to the total number of meitic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) o isp-1 (B), and left untreated or exposed to 1200 J/m2 UVB. Apoptotic cells were counted 12, 24 and 36 hours after exposure Data information: Data are presented as mean ± SEM, N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOV Tukey"s multiple comparisonsReal Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented a mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsReal Time PCR gene expression analysis of egl- genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated wit UV Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsReal Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treated with gamma radiatio Data information: Data are presented as mean ± SE N= * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonsReal Time PCR gene expression analysis o ced-1 genes, performed in wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 and left untreated or treate with UV Data information: Data are presented as mean ± SE N= F). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs untreated; # p < 0.05 and ## p < 0.01 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s mu comparisons test


Figure 4Representative fluorescence pictures and quantification ( below the fluorescence pictures ) of dissected adult gonads relative to the number of mitotic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 and stained with DAPI ( blue ) and either anti - PARP - 1 ( red ) ( A Scale bar : 20µ ( A Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and conditionRepresentative fluorescence pictures and quantification ( below the fluorescence pictures ) of dissected adult gonads relative to the number of mitotic cells from wild - type animals fed bacteria transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 and stained with DAPI ( blue and eithe anti - RPA - 1 ( green ) and anti - RAD - 51 ( red ) antibodies and treated with gamma radiation ( 125 Gy ) ( B ) ( untreated animals displayed no staining of RPA - 1 and RAD - 51 , not shown ) 15µm ( B Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and conditionQuantification of mitotic cells in wild - type animals fed as ( A ) and left untreated or treated with gamma radiation 125 G Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and conditionQuantification of mitotic cells in wild - type animals fed as ( A ) and left untreated or treate with 1200 J / m2 UV Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate and

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

Figure 4Representative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue) and either anti-PARP-1 (red) (A Scale bar: 20µ (A Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and conditionRepresentative fluorescence pictures and quantification (below the fluorescence pictures) of dissected adult gonads relative to the number of mitotic cells from wild-type animals fed bacteria transformed with either vector expressing dsRNA against gfp (con) or frh-1 and stained with DAPI (blue and eithe anti-RPA-1 (green) and anti-RAD-51 (red) antibodies and treated with gamma radiation (125 Gy) (B) (untreated animals displayed no staining of RPA-1 and RAD-51, not shown) 15µm (B Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and conditionQuantification of mitotic cells in wild-type animals fed as (A) and left untreated or treated with gamma radiation 125 G Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and conditionQuantification of mitotic cells in wild-type animals fed as (A) and left untreated or treate with 1200 J/m2 UV Data information: Data are presented as mean ± SD. For each panel N=2, 8-10 worms per replicate and condition


Figure 5A , B Quantification to the number of mitotic cells of dissected gonads from wild - type animals stained with either anti - phosphohistone H3 ( PH3 ) ( A ) , anti - CDK - 1 ( CDK - 1 ) ( B ) transformed with either empty vector ( con ) or vector - expressing dsRNA against frh - 1 ( frh - 1 ) or isp - 1 ( isp - 1 ) and treated with gamma radiation 125 Gy . Quantification is expressed as fold changes of positive stained cells treated over the positive untreated cells Data information : Data are presented as mean ± SD ( A , B N = 2 ( A , B * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . One - way ANOVA Tukey " s multiple comparisons testFertilit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated with gamma radiatio at the indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified 2 - wa 2 - way ANOVA Tukey " s multiple compa ( C - H ) fecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated wit UV at the indicated doses Data information : Data are presented a mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple compaFertilit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated wit Hydroxyure at the indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple comparisonsfecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated with gamma radiatio at the indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple compafecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , and left untreated or treated wit UV at the indicated doses Data information : Data are presente as mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple compafecundit quantification of wild - type strain fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , an left untreated or treated wit Hydroxyure at the indicated doses Data information : Data are presente as mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

Figure 5A, B Quantification to the number of mitotic cells of dissected gonads from wild-type animals stained with either anti-phosphohistone H3 (PH3) (A), anti-CDK-1 (CDK-1) (B) transformed with either empty vector (con) or vector-expressing dsRNA against frh-1 (frh-1) or isp-1 (isp-1) and treated with gamma radiation 125 Gy. Quantification is expressed as fold changes of positive stained cells treated over the positive untreated cells Data information: Data are presented as mean ± SD (A, B N=2 (A, B * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. One-way ANOVA Tukey"s multiple comparisons testFertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiatio at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified 2-wa 2-way ANOVA Tukey"s multiple compa (C-H)fecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit UV at the indicated doses Data information: Data are presented a mean ± SE ), N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compaFertilit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple comparisonsfecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated with gamma radiatio at the indicated doses Data information: Data are presented a mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compafecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, and left untreated or treated wit UV at the indicated doses Data information: Data are presente as mean ± SE ), N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compafecundit quantification of wild-type strain fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1, an left untreated or treated wit Hydroxyure at the indicated doses Data information: Data are presente as mean ± SE N= * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 vs untreated; # P < 0.05, ### P < 0.001, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified , B), 2-way ANOVA Tukey"s multiple compa


Figure 6A Quantification of BRD - 1 protein and representative western blot from wild - type worms fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 Bar graphs are mean ± SEM Data information : Data are presented as mean ± SD N = 8 - 1 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specifiedB Quantification of mitotic nuclei in single gonad arm of wild - type ( WT ) , brc - 1 ( tm1145 ) and brd - 1 ( gk297 ) strains fed as in ( A ) Data information : Data are presented as mean ± SD N = ) , 8 - 1 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specifiedQuantification of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) and brd - 1 ( gk297 ) strains fed as in ( A ) and left untreated ( C Bar graphs are mean ± SEM . Data information Data are presented a mean ± SEM N = 8 - 10 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple comparisonQuantification of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) and brd - 1 ( gk297 ) strains fed as in ( A ) an treated with ionizing radiation 125 Gy ( IR ) ( D ) . Bar graphs are mean ± SEM . Data information : Data are presented a mean ± SEM N = 8 - 10 worms per replicate and condition , * P < 0 . 05 , * * * P < 0 . 001 and * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 and # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Figure 6A Quantification of BRD-1 protein and representative western blot from wild-type worms fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1 Bar graphs are mean ± SEM Data information: Data are presented as mean ± SD N= 8-1 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specifiedB Quantification of mitotic nuclei in single gonad arm of wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) Data information: Data are presented as mean ± SD N= ), 8-1 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specifiedQuantification of apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) and left untreated (C Bar graphs are mean ± SEM. Data information Data are presented a mean ± SEM N= 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparisonQuantification of apoptotic corpse in wild-type (WT), brc-1(tm1145) and brd-1(gk297) strains fed as in (A) an treated with ionizing radiation 125 Gy (IR) (D). Bar graphs are mean ± SEM. Data information: Data are presented a mean ± SEM N= 8-10 worms per replicate and condition, * P < 0.05, *** P < 0.001 and **** P < 0.0001 vs untreated; ### P<0.001 and #### P < 0.0001 vs con. Not significant if nothing is specified. 2-way ANOVA Tukey"s multiple comparison


Figure 7Kaplan - Meier survival curves of wild - type ( WT ) and , ced - 9 ( n1653ts strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an ) , brc - 1 ( tm1145 ) ( B strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an brd - 1 ( gk297 ) ( C strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an glp - 1 ( e2141 ) ( D strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type ( WT ) an glp4 ( bn2 ) ( E ) , strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1Kaplan - Meier survival curves of wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp - 1 ( F and left untreated or treated with 600 , 1200 or 2400 J / m2 UVBKaplan - Meier survival curves of wild - type animals fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA agains isp - 1 ( G ) and left untreated or treated with 600 , 1200 or 2400 J / m2

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

Figure 7Kaplan-Meier survival curves of wild-type (WT) and, ced-9(n1653ts strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an ), brc-1(tm1145) (B strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an brd-1(gk297) (C strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an glp-1(e2141) (D strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type (WT) an glp4(bn2) (E), strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against frh-1 or isp-1Kaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1 (F and left untreated or treated with 600, 1200 or 2400 J/m2 UVBKaplan-Meier survival curves of wild-type animals fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA agains isp-1 (G) and left untreated or treated with 600, 1200 or 2400 J/m2 UVB


Figure 8A Kaplan - Meier survival curves of wild - type ( WT ) and , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed bacteria transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp - 1B Quantification of apoptotic corpses in wild - type ( WT ) and , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed as ( A ) and left untreated or treated with 600 J / m2 UVB . Data information : Data are presented as mean ± SEM , N = 3 , 9 - 10 worms per replicate and condition * * P < 0 . 01 vs con ; # # P < 0 . 01 vs WT ; ooo P < 0 . 00 and oooo P < 0 . 0001 vs untreated . Not significant if nothing is specified . 2 - way ANOVA Tukey ' s multiple comparisons test . C

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

Figure 8A Kaplan-Meier survival curves of wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed bacteria transformed with either empty-vector (con) or with vector expressing dsRNA against isp-1B Quantification of apoptotic corpses in wild-type (WT) and, rrf-1(pk1417), ppw-1(pk2505) strains fed as (A) and left untreated or treated with 600 J/m2 UVB. Data information: Data are presented as mean ± SEM, N=3, 9-10 worms per replicate and condition ** P < 0.01 vs con; ## P < 0.01 vs WT; ooo P < 0.00 and oooo P < 0.0001 vs untreated . Not significant if nothing is specified. 2-way ANOVA Tukey's multiple comparisons test. C


Figure 1Western blot of the indicated proteins in T - inducible PSCs stimulated as indicated for 24 hr . GAPDH was used as a loading control .

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Figure 1Western blot of the indicated proteins in T-inducible PSCs stimulated as indicated for 24 hr. GAPDH was used as a loading control.


Figure 2Heatmap derived from mean - centered expression values ( log2 scale ) of selected key pluripotency genes . See also Fig EV2C , Dataset EV1 for all the clustered DEGs .

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Figure 2Heatmap derived from mean-centered expression values (log2 scale) of selected key pluripotency genes. See also Fig EV2C, Dataset EV1 for all the clustered DEGs.


Figure 3ChIP - seq tracks of T ( in red ; input is in blue ) around the Oct4 ( 7 . 5 kb ) . DE , distal enhancer ; PE , proximal enhancer . The T - binding peak around the Oct4 - DE is indicated ( arrowhead ) . Right : ChIP - qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E . NC , negative control ( the locus without T binding ) . The enrichment is indicated by the percent input with SDs ( three independent experiments with two technical replicates ) . Luciferase assay with T - binding elements ( TBEs ) around the indicated loci as shown in ( A ) and Fig EV3E . Relative luciferase activities of sorted cell fractions are shown with SDs . ( three independent experiments with two technical replicates ) .

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Figure 3ChIP-seq tracks of T (in red; input is in blue) around the Oct4 (7.5 kb). DE, distal enhancer; PE, proximal enhancer. The T-binding peak around the Oct4-DE is indicated (arrowhead). Right: ChIP-qPCR for the enrichment of T on the DE and the regions indicated in Fig EV3E. NC, negative control (the locus without T binding). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).Luciferase assay with T-binding elements (TBEs) around the indicated loci as shown in (A) and Fig EV3E. Relative luciferase activities of sorted cell fractions are shown with SDs. (three independent experiments with two technical replicates).


Figure 4Left : A representative confocal image of a mouse embryo at E7 . 0 bearing T - GFP and Blimp1 - RFP transgenic reporters . Scale bar : 50um . Arrowheads indicate Blimp1 - positive nascent germ cells in the posterior proximal epiblast . Right : FACS analysis of dissociated mouse embryos without visceral endoderm . Expression of the indicated genes measured by qPCR in FACS - sorted cells ( E ) derived from embryos at different developmental stages ( E7 . 0 - 7 . 5 ) . Ten FACS - sorted cells in different cell fractions ( E ) were used for each individual qPCR analysis with two technical replicates . The ∆ Ct values from the average Ct values of Arbp and Ppia are shown ( n ≥ 3 , log2 scale ) . P values were calculated by two - tailed unpaired t - test .

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Figure 4Left: A representative confocal image of a mouse embryo at E7.0 bearing T-GFP and Blimp1-RFP transgenic reporters. Scale bar: 50um. Arrowheads indicate Blimp1-positive nascent germ cells in the posterior proximal epiblast. Right: FACS analysis of dissociated mouse embryos without visceral endoderm.Expression of the indicated genes measured by qPCR in FACS-sorted cells (E) derived from embryos at different developmental stages (E7.0-7.5). Ten FACS-sorted cells in different cell fractions (E) were used for each individual qPCR analysis with two technical replicates. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (n≥3, log2 scale). P values were calculated by two-tailed unpaired t-test.


Figure 5ChIP - seq tracks of T ( in red ; input is in blue ) and core pluripotency factors ( in green ) around the Blimp1 ( 100 kb ) and Prdm14 ( 100 kb ) . The co - bound locus ( L # 1 - 4 ) and the locus exclusively bound by core pluripotency factors but not T ( L # 5 ) are indicated by arrow . NC , negative control ( the locus without T binding ) . ChIP - qPCR for the enrichment of the indicated factors on the loci ( L # 1 - 5 ) indicated in ( A ) . The enrichment is indicated by the percent input with SDs ( three independent experiments with two technical replicates ) . Luciferase assay with the specific sequence of the locus bound by T around L # 1 and L # 3 as shown in ( A ) . Relative luciferase activities of low T - inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs . ( three independent experiments with two technical replicates ) . Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines . The ∆ Ct values from the average Ct values of Arbp and Ppia are shown ( log2 scale , the mean value of two independent experiments with two technical replicates ) . Top : Oct4 - GFP ( green ) reporter expression ( left ) , and Blimp1 - RFP ( red ) and Oct4 - GFP ( BROG ) reporter expression ( right ) in the gonads of E13 . 5 embryos generated by 4N complementation with the indicated ESC lines . Scale bar : 100um . Bottom : percentage of Oct4 - GFP reporter - positive PGCs ( left ) and BROG reporter - positive PGCs ( right ) measured by FACS analysis . Blue circles , red squares , and green triangles represent the percentage of gonadal PGCs in the first , second , and third independent mutant line , respectively , of the examined locus ( L # 1 - 5 ) .

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Figure 5ChIP-seq tracks of T (in red; input is in blue) and core pluripotency factors (in green) around the Blimp1 (100 kb) and Prdm14 (100 kb). The co-bound locus (L#1-4) and the locus exclusively bound by core pluripotency factors but not T (L#5) are indicated by arrow. NC, negative control (the locus without T binding).ChIP-qPCR for the enrichment of the indicated factors on the loci (L#1-5) indicated in (A). The enrichment is indicated by the percent input with SDs (three independent experiments with two technical replicates).Luciferase assay with the specific sequence of the locus bound by T around L#1 and L#3 as shown in (A). Relative luciferase activities of low T-inducible ZHBTc4 ESCs stimulated as indicated are shown with SDs. (three independent experiments with two technical replicates).Expression of Blimp1 and Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines. The ∆Ct values from the average Ct values of Arbp and Ppia are shown (log2 scale, the mean value of two independent experiments with two technical replicates).Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5).


Figure 1High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry . Lack of human SDF - 1α release from cultured SW1417 CRC cells , as measured by ELISA , whereas human control 1BR3 . G fibroblasts express high SDF - 1α levels , after 48h or 72h of growth in culture . ( N = 2 experiment in duplicate ) Nanoconjugate internalization in CXCR4 overexpressing ( CXCR4 + ) SW1417 CRC cells after 1 hour exposure at 1µM , as measured by fluorescence emission using flow cytometry . ( N = 3 experiments in duplicate ) Significant difference at p = 0 . 002 between T22 - GFP - H6 - FdU and AMD3100 + T22 - GFP - H6 - FdU groupsIntracellular trafficking of T22 - GFP - H6 - FdU in CXCR4 + SW1417 cells by confocal microscopy after exposure at 1µM for 24 h . The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye ( CellMask ™ ) , whereas cell nucleus was stained in blue with hoescht . The insets show detail of the intracellular localization of nanostructured , fluorescent entities , in an isosurface representation within a three - dimensional volumetric x - y - z data fieldLinearized T22 - GFP - H6 - FdU dose - response trend line representation compared with unconjugated free oligo - FdU exposure . Antitumor effect was measured as CXCR4 + SW1417 cell viability by MTT after 72 h exposure as the described concentrations . ( N = 3 experiments in duplicate ) Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22 - GFP - H6 - FdU for 48 h , as compared to T22 - GFP - H6 or free oligo - FdU . ( N = 3 experiments in duplicate ; Scale bar , 100µm ) .

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Figure 1High and constitutive expression of CXCR4 in the membrane of SW1417 CRC cells as measured by flow cytometry.Lack of human SDF-1α release from cultured SW1417 CRC cells, as measured by ELISA, whereas human control 1BR3.G fibroblasts express high SDF-1α levels, after 48h or 72h of growth in culture. (N=2 experiment in duplicate)Nanoconjugate internalization in CXCR4 overexpressing (CXCR4+) SW1417 CRC cells after 1 hour exposure at 1µM, as measured by fluorescence emission using flow cytometry. (N=3 experiments in duplicate) Significant difference at p= 0.002 between T22-GFP-H6-FdU and AMD3100+T22-GFP-H6-FdU groupsIntracellular trafficking of T22-GFP-H6-FdU in CXCR4+ SW1417 cells by confocal microscopy after exposure at 1µM for 24 h. The green staining corresponds to GFP containing nanoconjugates and the red staining corresponds to plasma cell membranes stained with a red dye (CellMask™), whereas cell nucleus was stained in blue with hoescht. The insets show detail of the intracellular localization of nanostructured, fluorescent entities, in an isosurface representation within a three-dimensional volumetric x-y-z data fieldLinearized T22-GFP-H6-FdU dose-response trend line representation compared with unconjugated free oligo-FdU exposure. Antitumor effect was measured as CXCR4+ SW1417 cell viability by MTT after 72 h exposure as the described concentrations. (N=3 experiments in duplicate)Reduction of cell viability determined by optical microscope images of SW1417 cells exposed to 1µM T22-GFP-H6-FdU for 48 h, as compared to T22-GFP-H6 or free oligo-FdU. (N=3 experiments in duplicate; Scale bar, 100µm).


Figure 2Selective T22 - GFP - H6 - FdU nanoconjugate biodistribution in subcutaneous CXCR4 + SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose , as measured by fluorescence emission using IVIS Spectrum 200 ( N = 5 / group ) . Biodistribution is similar to that achieved by the T22 - GFP - H6 targeting vector and undetectable after Buffer or free oligo - FdU treatment . ( N = 5 mice / group ) Co - localization ( yellow merged ) of the T22 - GFP - H6 - FdU ( green ) and the CXCR4 receptor ( red ) and release of T22 - GFP - H6 - FdU into the cytosol in CXCR4 + tumor cells 5h after a 100ug dose of nanoconjugate , as measured by dual anti - GFP / anti - CXCR4 immunofluorescence ( IF ) . DAPI ( blue nuclear staining ) . Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area ( A ) ± s . e . m ( µm2 ) ( N = 10 , 2 tumor fields x 5 mice ; 200X ) . Note the significant ( P = 0 . 003 ) increase in the area occupied by the green dots ( nanoconjugate realeased to the cell cytosol ) in T22 - GFP - H6 - FdU - treated tumors , compared to free oligo - FdU - treated control tissues . Scale bar , 50 µm . Administration of the CXCR4 antagonist AMD3100 completely blocks T22 - GFP - H6 - FdU tumor biodistribution , as measured by fluorescence emission . Fluorescence is not detected in Buffer or free oligo - FdU controls ( N = 5 tumor fields / group ) . The uptake of T22 - GFP - H6 - FdU observed in CXCR4 + SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration , as quantified using the anti - GFP IHC H - score . ( N = 5 tumor fields / group ) . Representative images of T22 - GFP - H6 - FdU uptake and AMD3100 competition by anti - GFP inmunostaining , which quantitation is reported in panel E . Scale bar , 50

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Figure 2Selective T22-GFP-H6-FdU nanoconjugate biodistribution in subcutaneous CXCR4+ SW1417 CRC tumor tissue 5h after a 100µg single intravenous dose, as measured by fluorescence emission using IVIS Spectrum 200 (N=5/group). Biodistribution is similar to that achieved by the T22-GFP-H6 targeting vector and undetectable after Buffer or free oligo-FdU treatment. (N=5 mice/group)Co-localization (yellow merged) of the T22-GFP-H6-FdU (green) and the CXCR4 receptor (red) and release of T22-GFP-H6-FdU into the cytosol in CXCR4+ tumor cells 5h after a 100ug dose of nanoconjugate, as measured by dual anti-GFP/anti-CXCR4 immunofluorescence (IF). DAPI (blue nuclear staining). Fluorescence emission was measured in the green and red channels using the Image J software and expressed as mean Area (A)±s.e.m (µm2) (N=10, 2 tumor fields x 5 mice; 200X). Note the significant (P=0.003) increase in the area occupied by the green dots (nanoconjugate realeased to the cell cytosol) in T22-GFP-H6-FdU-treated tumors, compared to free oligo-FdU-treated control tissues. Scale bar, 50 µm.Administration of the CXCR4 antagonist AMD3100 completely blocks T22-GFP-H6-FdU tumor biodistribution, as measured by fluorescence emission. Fluorescence is not detected in Buffer or free oligo-FdU controls (N=5 tumor fields/group).The uptake of T22-GFP-H6-FdU observed in CXCR4+ SW1417 tumor tissues is almost completely blocked by prior AMD3100 administration, as quantified using the anti-GFP IHC H-score. (N=5 tumor fields/group).Representative images of T22-GFP-H6-FdU uptake and AMD3100 competition by anti-GFP inmunostaining, which quantitation is reported in panel E. Scale bar, 50 µm


Figure 3Representative images of CXCR4 overexpression in subcutaneous tumor tissue , showing similar CXCR4 levels among compared groups ( N = 5 / group ; Buffer , T22 - GFP - H6 - FdU , T22 - GFP - H6 and free oligo - FdU ) before treatment ( upper panels ) . Representative images of DNA double - strand break induction and caspase 3 activation ( measured with anti - ɣH2AX or anti - cleaved caspase 3 by IHC ) 5h post - administration ( middle panels ) . Apoptotic induction ( Hoescht staining , 24h post - administration , lower panels ) . Note the higher number of cells positive for DSBs , Caspase 3 activation and apoptosis induction in the T22 - GFP - H6 - FdU as compared to free oligo - FdU . Black or white arrows indicate dead cells . Quantitation of the number of cells containing DSBs or active Caspase - 3 in IHC - stained tumor sections 5h post - treatment and the number of condensated or disaggregated nuclei ( by Hoescht staining ) 24h post - treatment in tumor sections of 10 high - powder fields ( 400x magnification ) using the Cell D software . ( N = 50 ; 10 tumor fields / mice ; 5 mice / group ) . Data expressed as mean ± s . e . m .

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Figure 3Representative images of CXCR4 overexpression in subcutaneous tumor tissue, showing similar CXCR4 levels among compared groups (N=5/group; Buffer, T22-GFP-H6-FdU, T22-GFP-H6 and free oligo-FdU) before treatment (upper panels). Representative images of DNA double-strand break induction and caspase 3 activation (measured with anti-ɣH2AX or anti-cleaved caspase 3 by IHC) 5h post-administration (middle panels). Apoptotic induction (Hoescht staining, 24h post-administration, lower panels). Note the higher number of cells positive for DSBs, Caspase 3 activation and apoptosis induction in the T22-GFP-H6-FdU as compared to free oligo-FdU. Black or white arrows indicate dead cells. Quantitation of the number of cells containing DSBs or active Caspase-3 in IHC-stained tumor sections 5h post-treatment and the number of condensated or disaggregated nuclei (by Hoescht staining) 24h post-treatment in tumor sections of 10 high-powder fields (400x magnification) using the Cell D software. (N=50; 10 tumor fields/mice; 5 mice/group). Data expressed as mean±s.e.m.


Figure 4T22 - GFP - H6 - FdU depletes CXCR4 + cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration . Note the reduction in CXCR4 + cell fraction in the tumor 24h after injection , their almost complete elimination at 48h and the re - emergence of CXCR4 + cells 72h post - administration , using anti - CXCR4 IHC . In contrast , the CXCR4 + cancer cell fraction ( CXCR4 + CCF ) in tumor tissue remains constant along time after free oligo - FdU treatment . The three day time - lapse for CXCR4 + tumor cell re - appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect . ( N = 5 : 5 mice / group ; 1 samples / mouse ) . Scale bar , 50 µm . Data expressed as mean ± s . e . mSignificant reduction in the number of spheroid formed ( C . optical microscope ) and their bioluminescence emission ( D , IVIS Spectrum 200 ) , generated by 1x106 disaggregated cells ( cultured in stem cell conditioned media and low - adhesion plates ) , obtained from CXCR4 + luciferase + SW1417 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . D . Quantitation of the bioluminescent signal ( BLI ) expressed as Average Radiant Intensity , obtained using the IVIS spectrum 200 equipment ( N = 2 plates / group ) .

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Figure 4T22-GFP-H6-FdU depletes CXCR4+ cancer cells from SW1417 CRC tumor tissue after a 100 µg single dose administration. Note the reduction in CXCR4+ cell fraction in the tumor 24h after injection, their almost complete elimination at 48h and the re-emergence of CXCR4+ cells 72h post-administration, using anti-CXCR4 IHC. In contrast, the CXCR4+ cancer cell fraction (CXCR4+ CCF) in tumor tissue remains constant along time after free oligo-FdU treatment. The three day time-lapse for CXCR4+ tumor cell re-appearance defines the dosage interval used in a repeated dose schedule of nanoconjugate administration in the experiments to evaluate its antimetastatic effect. (N=5: 5 mice/group; 1 samples/mouse). Scale bar, 50 µm. Data expressed as mean±s.e.mSignificant reduction in the number of spheroid formed (C. optical microscope) and their bioluminescence emission (D, IVIS Spectrum 200), generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates), obtained from CXCR4+ luciferase+ SW1417 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. D. Quantitation of the bioluminescent signal (BLI) expressed as Average Radiant Intensity, obtained using the IVIS spectrum 200 equipment (N=2 plates/group).


Figure 5Reduction in the number of formed spheroids ( optical microscope ) generated by 1x106 disaggregated cells ( cultured in stem cell conditioned media and low - adhesion plates ) obtained from CXCR4 + M5 subcutaneous tumors , 24h after 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . ( N = 8 ; 2 mice / group ; 4 plates / mouse ) . ( Scale bar , 100µm ) . Reduction in tumor re - initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice ( N = 4 tumors / group ) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Free oligo - FdU - treated or Buffer - treated mice . Recording of the number and size of positive tumors . ( N = 4 ; 2 mice / group , 2 injection points / mouse ) . T22 - GFP - H6 - FdU - induced reduction in the number of intravasated tumor emboli in peri - tumoral vessels of the M5 - orthotopic primary tumor ( E . optical images ; F . emboli number quantitation ) and reduction of CXCR4 expression in these emboli ( G ) , treated 7 days after tumor cell implantation with 100µg T22 - GFP - H6 - FdU intravenous doses , for 2 consecutive days , as compared to Buffer - treated or free oligo - FdU - treated mice . Tumor emboli counting in 10 high powder field at 200x magnification in H & E stained sections from each tumor . ( N = 5 / group ) .

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Figure 5Reduction in the number of formed spheroids (optical microscope) generated by 1x106 disaggregated cells (cultured in stem cell conditioned media and low-adhesion plates) obtained from CXCR4+ M5 subcutaneous tumors, 24h after 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. (N=8; 2 mice/group; 4 plates/mouse). (Scale bar, 100µm).Reduction in tumor re-initiation capacity after subcutaneous inoculation of 5x106 cells in NSG mice (N=4 tumors/group) derived from disaggregated tumor cells obtained from SC tumors 10 days after administration of 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days , as compared to Free oligo-FdU-treated or Buffer-treated mice. Recording of the number and size of positive tumors. (N=4; 2 mice/group, 2 injection points/mouse).T22-GFP-H6-FdU-induced reduction in the number of intravasated tumor emboli in peri-tumoral vessels of the M5-orthotopic primary tumor (E. optical images; F. emboli number quantitation) and reduction of CXCR4 expression in these emboli (G), treated 7 days after tumor cell implantation with 100µg T22-GFP-H6-FdU intravenous doses, for 2 consecutive days, as compared to Buffer-treated or free oligo-FdU-treated mice. Tumor emboli counting in 10 high powder field at 200x magnification in H&E stained sections from each tumor. (N=5/group).


Figure 6T22 - GFP - H6 - FdU prevents metastases in the CXCR4 + patient - derived M5 model by potently reducing the total and mean number of liver , lung and peritoneal mets , as recorded in H & E stained histology sections at the end of treatment , in comparison to free oligo - FdU o Buffer - treatment . In contrast , the number of LN mets is not reduced after T22 - GFP - H6 - FdU or free oligo - FdU administration ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group and N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e . m . T22 - GFP - H6 - FdU induces a higher reduction of CXCR4 + cancer cell fraction ( CXCR4 + CCF ) in liver , lung and peritoneal metastatic tissue , at the end of treatment , than free oligo - FdU , as measured by anti - CXCR4 IHC . In contrast , T22 - GFP - H6 - FdU or free oligo - FdU does not reduce the CXCR4 + CCF in LN mets or primary tumor tissue after therapy . ( N = 6 mice per Buffer group ; N = 7 mice per Free oligo - FdU group and N = 8 mice per T22 - GFP - H6 - FdU group ; 3 samples / mouse ) . Data expressed as mean ± s . e . mRepresentative CXCR4 IHC images of the reduction in CXCR4 + CCF induced by T22 - GFP - H6 - FdU ( or its absence in free oligo - FdU mice ) at the end of treatment , in the M5 patient - derived CRC model , which quantitation is reported in panel B . In the M5 model , the highest reduction in foci number and size occurs in liver metastases , which show the highest reduction in CXCR4 + CCF .

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

Figure 6T22-GFP-H6-FdU prevents metastases in the CXCR4+ patient-derived M5 model by potently reducing the total and mean number of liver, lung and peritoneal mets, as recorded in H&E stained histology sections at the end of treatment, in comparison to free oligo-FdU o Buffer-treatment. In contrast, the number of LN mets is not reduced after T22-GFP-H6-FdU or free oligo-FdU administration (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.m.T22-GFP-H6-FdU induces a higher reduction of CXCR4+ cancer cell fraction (CXCR4+ CCF) in liver, lung and peritoneal metastatic tissue, at the end of treatment, than free oligo-FdU, as measured by anti-CXCR4 IHC. In contrast, T22-GFP-H6-FdU or free oligo-FdU does not reduce the CXCR4+ CCF in LN mets or primary tumor tissue after therapy. (N=6 mice per Buffer group; N=7 mice per Free oligo-FdU group and N=8 mice per T22-GFP-H6-FdU group; 3 samples/mouse). Data expressed as mean±s.e.mRepresentative CXCR4 IHC images of the reduction in CXCR4+ CCF induced by T22-GFP-H6-FdU (or its absence in free oligo-FdU mice) at the end of treatment, in the M5 patient-derived CRC model, which quantitation is reported in panel B. In the M5 model, the highest reduction in foci number and size occurs in liver metastases, which show the highest reduction in CXCR4+ CCF.