( C ) Still frames from time - lapse recordings of an expressing a myosin - II probe ( Sqh : : GFP ) . were mounted vertically to image transverse cross - section using two - photon microscopy . At onset of gastrulation , myosin - II localized to ring structures representing leading edge of cellularization front ( lower arrow ) . During ventral furrow formation ( torques open rectangle ) , myosin - II accumulated at apical side ( upper arrow ) of that invaginate basal pool was progressively depleted . Scale bar , 40 µm

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

(C) Still frames from time-lapse recordings of an embryo expressing a myosin-II probe (Sqh::GFP). Embryos were mounted vertically to image the transverse cross-section using two-photon microscopy. At the onset of gastrulation, myosin-II localized to ring structures representing the leading edge of the cellularization front (lower arrow). During ventral furrow formation (torques open rectangle), myosin-II accumulated at the apical side (upper arrow) of the cells that invaginate and the basal pool was progressively depleted. Scale bar, 40 µm


( D ) of basal myosin - II ( N = 3 ) in ectodermal mesodermal . In mesoderm , basal myosin - II decreasd 5 - fold over course of internalization , while in ectoderm only ~ 3 - fold . Additionally , myosin - II depletion appeared to be accelerated in mesodermal as indicated by steeper slope of polynomial fit ( 3rd degree with r2Ectoderm : 0 . 98 r2Mesoderm : 0 . 87 ) . Note logarithmic y - axis

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

(D) Quantification of basal myosin-II levels (N=3 embryos) in ectodermal and mesodermal cells. In the mesoderm, basal myosin-II levels decreasd 5-fold over the course of internalization, while in the ectoderm only ~3-fold. Additionally, myosin-II depletion appeared to be accelerated in the mesodermal cells as indicated by the steeper slope of the polynomial fit (3rd degree with r2Ectoderm: 0.98 and r2Mesoderm: 0.87). Note the logarithmic y-axis


( E - H ) at end of cellularization co - expressing RhoGEF2 - CRY2 ; CIBN : : pmGFP , Sqh : : mCherry were mounted with dorsal epithelium facing objective a region of interest ( red dashed ) was defined to specifically illuminate base using two - photon microscopy . A representative ( N = 3 ) showing basal myosin - II from top view ( sum - of - slice projection of 6 focal planes ) at initial stage of experiment ( E ) 6 min after activation ( F ) . Z - projection of same showing cross section of activated region ( AR ) ( G ) non - activated region ( NR ) ( H ) with superimposed plasma signal ( CIBN : : GFPpm in magenta ) recorded immediately after final Sqh : : mCherry acquisition ( displayed in ) . contrast has been adjusted to better visualize distribution of myosin - II along apicobasal axis in respective of tissue . Scale bars , 20 µm

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

(E-H) Embryos at the end of cellularization co-expressing RhoGEF2-CRY2; CIBN::pmGFP, and Sqh::mCherry were mounted with the dorsal epithelium facing the objective and a region of interest (red dashed line) was defined to specifically illuminate the cell base using two-photon microscopy. A representative embryo (N=3) showing basal myosin-II from top view (sum-of-slice projection of 6 focal planes) at the initial stage of the experiment (E) and 6 min after activation (F). Z-projection of the same embryo showing the cross section of the activated region (AR) (G) and non-activated region (NR) (H) with the superimposed plasma membrane signal (CIBN::GFPpm in magenta) recorded immediately after the final Sqh::mCherry acquisition (displayed in white). The contrast has been adjusted to better visualize the distribution of myosin-II along the apicobasal axis in the respective area of the tissue. Scale bars, 20 µm


( I ) of myosin - II ( N = 3 ) within region of activation ( AR , red ) relative to non - activated region ( NR , green ) over course of 6 min . y - axis represents fold - change of myosin - II signal in activated region versus non - activated region . A polynomial function ( 2nd degree , r2AR = 0 . 88 ) was fitted to data ( straight ) indicating level of saturation that is reached at about 1 . 6 - fold change of myosin - II in activated after 6 min

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

(I) Quantification of myosin-II levels (N=3) within the region of activation (AR, red) relative to the non-activated region (NR, green) over the course of 6 min. The y-axis represents the fold-change of myosin-II signal intensity in the activated region versus non-activated region. A polynomial function (2nd degree, r2AR = 0.88) was fitted to the data (straight line) indicating the level of saturation that is reached at about 1.6-fold change of myosin-II in the activated area after 6 min


( J of extent of signal spreading in z - dimension ( apico - basla ) of basal myosin - II . Myosin - II signal was plotted along apicobasal axis a Gaussian curve fitted to basal peak . width of Gaussian fit was used as a parameter to estimate myosin - II signal spreading in z - dimension , which was limited to 4 µm ( N = 3 ) . In each box plot , central mark , bottom , top edge of each box indicates median , 25th 75th percentile , respectively . Whiskers extent to most extreme data

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

(J Analysis of the extent of signal spreading in the z-dimension (apico-basla) of basal myosin-II. Myosin-II signal intensity was plotted along the apicobasal axis and a Gaussian curve fitted to the basal peak. The width of the Gaussian fit was used as a parameter to estimate myosin-II signal spreading in the z-dimension, which was limited to 4 µm (N=3). 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


( K ) of diameter of basal actomyosin rings in non - activated region ( NR , green ) activated region ( AR , red ) showing increased speed ( 4 . 8 - fold change ) extent of ring constrictions in photo - activated region ( n = > 41 ) . ring diameter was normalized to mean value of first time point . constriction speed was estimated by slope of a fitted linear function to data . ( L ) activation of base resulted in a compaction of tissue in activated ( AR ) compared to non - activated region ( NR ) . Actomyosin rings were counted normalized to analyzed to score ring density , which increased 1 . 35 times in activated region after 5 min of photo - activation , showing that were still interconnected at base that photo - activation did not affect tissue integrity ( N = 3 ; nNR : 431 , nAR : 302 ) . In each box plot , central mark , bottom , top edge of each box indicates median , 25th 75th percentile , respectively . Whiskers extent to most extreme data point

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

(K) Quantification of the diameter of basal actomyosin rings in the non-activated region (NR, green) and activated region (AR, red) showing increased speed (4.8-fold change) and extent of ring constrictions in the photo-activated region (n= >41 cells). The ring diameter was normalized to the mean value of the first time point. The constriction speed was estimated by the slope of a fitted linear function to the data. (L) Light activation of the cell base resulted in a compaction of the tissue in the activated (AR) compared to the non-activated region (NR). Actomyosin rings were counted and normalized to the analyzed area to score ring density, which increased 1.35 times in the activated region after 5 min of photo-activation, showing that cells were still interconnected at the base and that photo-activation did not affect tissue integrity (N = 3 embryos; nNR: 431, nAR: 302). 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


Ventral furrow formation was inhibited upon photo - stabilization of myosin - II at base . co - expressing optogenetic module myosin - II probe Sqh : : mCh were mounted with ventral tissue facing objective . A region of photo - activation ( two - photon λ = 950 nm ) was defined at base in anterior half of , whole - myosin - II signal was recorded ( confocal setup ; λ = 561 nm ) in an alternating fashion . Scale bars , 20 µm . ( A ) Top view showing apical myosin - II 10 min after initial activation . While in non - activated region have internalized disappeared from apical focal planes ( right ) , in activated region failed to internalize despite showing myosin - II accumulation at apical surface ( left ) . Red Blue dotted lines indicate location of cross - section panels displayed in ( C ) ( D ) . ( B ) of apical myosin - II demonstrate that defects in tissue invagination were not due to impaired apical myosin - II accumulation as average apical myosin - II level per did not significantly differ between non - activated ( NR ) activated region ( AR ) . values were normalized using median value of combined population in NR AR . ( N = 3 ; nNR : 94 , nAR : 78 ) . Significances ( by two - sample t - ) : n . s . indicates no statistically significant differences . In each box plot , central mark , bottom , top edge of each box indicates median , 25th 75th percentile , respectively . Whiskers extent to most extreme data point ' + ' symbol indicates an outlier . ( C

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

Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm. (A) Top view showing apical myosin-II 10 min after the initial activation. While cells in the non-activated region have internalized and disappeared from the apical focal planes (right), cells in the activated region failed to internalize despite showing myosin-II accumulation at the apical surface (left). Red and Blue dotted lines indicate the location of the cross-section panels displayed in (C) and (D). (B) Quantification of apical myosin-II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin-II accumulation as the average apical myosin-II level per cell did not significantly differ between the non-activated (NR) and activated region (AR). The intensity values were normalized using the median value of the combined cell population in the NR and AR. (N = 3 embryos; nNR: 94, nAR: 78). Significances (by two-sample t-Test): n.s. indicates no statistically significant differences. 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 and the '+' symbol indicates an outlier. (C


Ventral furrow formation was inhibited upon photo - stabilization of myosin - II at base . co - expressing optogenetic module myosin - II probe Sqh : : mCh were mounted with ventral tissue facing objective . A region of photo - activation ( two - photon λ = 950 nm ) was defined at base in anterior half of , whole - myosin - II signal was recorded ( confocal setup ; λ = 561 nm ) in an alternating fashion . Scale bars , 20 µm ( C , D ) z - projections ( transverse cross section ) of myosin - II time - lapse recordings of non - activated ( C ) activated region ( D ) . While in non - activated region internalized , resulting in a complete closure of furrow ( C ) , stabilization of basal myosin - II prevented invagination ( D ) . Scale bars , 20

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

Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm (C, D) z-projections (transverse cross section) of myosin-II time-lapse recordings of the non-activated (C) and activated region (D). While cells in the non-activated region internalized, resulting in a complete closure of the furrow (C), stabilization of basal myosin-II levels prevented invagination (D). Scale bars, 20 µm


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

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

(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.


expressing RhoGEF2 - CRY2 / CIBN : : GFPpm marker GAP43 : : mCherry were mounted with ventral epithelium facing objective . To analyze apical dynamics at each activation cycle , photo - activation time was limited to ~ 20 s was followed by acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . basal surface of anterior ( left ) half of was photo - activated . ( A ) Still frames of time lapse ( GAP43 : : mCherry ) recording are shown at initial time point ( top ) , after 6 . 5 min ( middle ) , after 13 min ( bottom ) of photo - activation . in non - activated region ( green ) underwent normal apical constriction . In activated region , two populations of were observed , one that constricted ( purple ) one that did not constrict ( red ) . in activated region that by end of experiment constricted to an smaller than half of their initial are considered as constricting . that constricted less were considered to be defective in apical constriction are referred to as non - constricting . Scale bar 20 µm .

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

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. (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.


expressing RhoGEF2 - CRY2 / CIBN : : GFPpm marker GAP43 : : mCherry were mounted with ventral epithelium facing objective . To analyze apical dynamics at each activation cycle , photo - activation time was limited to ~ 20 s was followed by acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . basal surface of anterior ( left ) half of was photo - activated . ( B ) show pulsatile behavior . Graphs illustrate of three representative in each category ( non - activated region : top ; activated region with constricting : middle ; activated region with non - constricting : bottom ) over course of one experiment . Yellow circles indicate position of automatically identified local maxima used for further analyses .

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

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.


expressing RhoGEF2 - CRY2 / CIBN : : GFPpm marker GAP43 : : mCherry were mounted with ventral epithelium facing objective . To analyze apical dynamics at each activation cycle , photo - activation time was limited to ~ 20 s was followed by acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . basal surface of anterior ( left ) half of was photo - activated . ( C , D ) ( C ) Diagram showing extent of ratchet - like constrictions . mean difference in apical between adjacent peaks ( relaxed surface ) is indicated by a red dot reflects extent to which shrink in a single . in non - activated region ( NR ) constricted on average more than 5 µm2 , which is characteristic for ratchet - like constriction . Non - constricting in activated region ( AR , non - con . ) displayed a mean difference in apical per of about 0 , reflecting non - ratchet constrictions meaning that pulsing constricted but relaxed subsequently back to their initial . Constricting in activated region ( AR , con . ) show an intermediate behavior with less efficient constrictions than in NR . Data points are significantly different as determined by ANOVA ( F ( 2 , 539 ) = 72 . 04 , p = 1 . 9e - 28 ) post - hoc Tukey . ( D ) pulsation period ( T ) . 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 D ) Red dots indicate mean values error bars represent standard deviation . More than 500 pulses ( nNR = 119 , nAR , non - con . = 128 , nAR , con = 295 ) of more than 100 were analyzed . Data information : In panels , * * * * indicates p ≤ 0 . 0001 , n . s . indicates no statistically significant differences according to two - sample t - .

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

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.


expressing RhoGEF2 - CRY2 / CIBN : : GFPpm marker GAP43 : : mCherry were mounted with ventral epithelium facing objective . To analyze apical dynamics at each activation cycle , photo - activation time was limited to ~ 20 s was followed by acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . basal surface of anterior ( left ) half of was photo - activated . ( E ) Diagram showing prevalence of in photo - activated region that constricted ( con . ) that did not constrict ( Non - con . ) . More than 4 / 5 of did not constrict to half of initial ( N = 5 ; n : 407 ; Cohen ' s D : d = 10 ) . In each box plot , central mark , bottom , top edge of each box indicates median , 25th 75th percentile , respectively . Whiskers extent to most extreme data point . Data information : In panels , * * * * indicates p ≤ 0 . 0001 , n . s . indicates no statistically significant differences according to two - sample t - .

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

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.


expressing RhoGEF2 - CRY2 / CIBN : : GFPpm marker GAP43 : : mCherry were mounted with ventral epithelium facing objective . To analyze apical dynamics at each activation cycle , photo - activation time was limited to ~ 20 s was followed by acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . basal surface of anterior ( left ) half of was photo - activated . ( F , G ) Graphs showing average apical ( F ) anisotropy ( G ) of in non - activated region ( green ) , in activated region that constricted ( purple ) that did not constrict ( red ) . straight indicates mean value at a given time - point , semi - transparent colored indicated respective standard deviation . in non - activated region constricted efficiently progressively acquired anisotropy . Constricting in activated region constricted slower in an isotropic manner . Non - constricting in activated region did not show net decrease in apical gain of anisotropy ( nNR = 34 , nAR , non - con . = 20 , nAR , con = 59 ) .

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

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).


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

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

(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


( A ) Localization of basal - specific optogenetic anchor CIBN - PatJ : : GFP - pm during ventral furrow formation . expressing CIBN - PatJ : : GFP - pm were imaged using two - photon excitation during ventral furrow formation . Transverse cross section ( z - projection ) at a stage when were already apically constricted elongated , ventral tissue already internalized .

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

(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.


co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY2 Sqh : : mCherry ( B , C were mounted with ventral tissue facing objective . After underwent apical constriction elongation basal volume of ventral was photo - activated using two - photon illumination 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 base as indicated by red arrowheads in activated region ( red ) . ( C ) Top view ( sum of slice of 4 µm - sized image stack ) showing basal myosin - II recruitment in photo - activated region ( red dashed square ) Data information : Scale bars , 20

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

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 µm


co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY an marker GAP43 : : mCherry ( D - I ) were mounted with ventral tissue facing objective . After underwent apical constriction elongation basal volume of ventral was photo - activated using two - photon illumination myosin - II signal was recorded in alternation ( D ) Cross section ( z - projection ) showing plasma ( E ) top view showing apical plasma at initial time point before photo - activation . At this stage , central mesodermal tissue has started to internalize were constricted at apical surface . In an alternating fashion , base of in anterior half of ( left ) was photo - activated for 100 sec GAP43 : : mCherry signal in entire field of view was recorded . furrow further ingressed closed completely 3 min ( F ) 6 min ( G ) after initial activation , respectively . In activated region , tissue failed to internalize to form a closed furrow both at 3 min ( H ) 6 min ( I ) after initial photo - activation . width of central tissue forming ventral furrow at initial point of experiment is 40 µm ( D ) . In non - activated region 6 min after initial activation width of tissue shrank to about 25 µm ( G ) , whereas it remained constant at 40 µm in activated region ( I ) . Data information : Scale bars , 20

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

Embryos 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


A , B of mitotic ( A ) meiotic ( B ) in distal germline in wild - type fed 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 condition , ( B ) N = 3 at least 10 worms for conditio * P < 0 . 05 * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

A, 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 comparisons


of dissected distal germline from wild - type stained either anti - phosphohistone H3 ( anti - PH3 ) ( C relative to total number of mitotic of wild - type Data informatio Data are presented a as mean ± S N = 2 at least 5 worms per replicate conditio * P < 0 . 05 * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

Quantification 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 comparisons


of dissected distal germline from wild - type stained eithe anti - CDK - 1 ( anti - CDK - 1 ( D relative to total number of mitotic of wild - type Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate conditio * P < 0 . 05 * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

Quantification 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 comparisons


of dissected distal germline from wild - type stained eithe 5 - ethynyl - 2 ′ - deoxyuridine ( EdU - green ) ( E ) relative to total number of mitotic of wild - type Data information : Data are presented a mean ± S N = 2 at least 5 worms per replicate conditio * P < 0 . 05 * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

Quantification of 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 comparisons


F Average number of apoptotic corpses in meiotic compartment relative to total number of meiotic 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 condition . * P < 0 . 05 * * * * P < 0 . 0001 vs con ( One - way ANOVA Tukey " s multiple

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

F 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


of apoptotic corpse in meiotic compartment of CED - 1 : : GF relative to total number of meiotic , fed transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated with gamma radiation 125 Gy ( A Data information : Data are presented as mean ± . For each panel at least N = 3 10 worms for replicate condition . * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification 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 comparisons


of apoptotic corpse in meiotic compartment of CED - 1 : : GF relative to total number of meiotic , fed transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated wit ) , UVC 400J / m2 ( B Data information : Data are presented as mean ± . For each panel at least N = 3 10 worms for replicate condition . * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification 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 comparisons


of apoptotic corpse in meiotic compartment o wild - type relative to total number of meiotic , fed transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated wit different doses of UVB ( C Data information : Data are presented as mean ± . For each panel at least N = 3 10 worms for replicate condition . * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification 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 comparisons


of apoptotic corpse in meiotic compartment of CED - 1 : : GF relative to total number of meiotic , fed transformed with either empty - vector ( con ) or vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated wit 100µM cisplatin Data information : Data are presented as mean ± . For each panel at least N = 3 10 worms for replicate condition . * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification 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


Time course of apoptotic corpses relative to total number of meitic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 ( A left untreated or exposed to 1200 J / m2 UVB . Apoptotic were counted 12 , 24 36 hours after exposure Data information : Data are presented as mean ± , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Time 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 comparisons


Time course of apoptotic corpses relative to total number of meitic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) o isp - 1 ( B ) , left untreated or exposed to 1200 J / m2 UVB . Apoptotic were counted 12 , 24 36 hours after exposure Data information : Data are presented as mean ± , N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOV Tukey " s multiple

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

Time 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 comparisons


Real Time PCR gene expression of egl - genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 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 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real 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 comparisons


Real Time PCR gene expression of egl - genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 left untreated or treated wit UV Data information : Data are presented as mean ± SE N = * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real 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 comparisons


Real Time PCR gene expression o ced - 1 genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 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 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Real 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 comparisons


Real Time PCR gene expression o ced - 1 genes , performed in wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 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 * * * * p < 0 . 0001 vs untreated ; # p < 0 . 05 # # p < 0 . 01 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s mu comparisons

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

Real 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


Representative pictures ( below pictures ) of dissected adult gonads relative to number of mitotic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 stained with DAPI ( blue ) 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

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

Representative 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 condition


Representative pictures ( below pictures ) of dissected adult gonads relative to number of mitotic from wild - type fed transformed with either vector expressing dsRNA against gfp ( con ) or frh - 1 stained with DAPI ( blue eithe anti - RPA - 1 ( green ) anti - RAD - 51 ( red ) antibodies treated with gamma radiation ( 125 Gy ) ( B ) ( untreated displayed no of RPA - 1 RAD - 51 , not shown ) 15µm ( B Data information : Data are presented as mean ± SD . For each panel N = 2 , 8 - 10 worms per replicate

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

Representative 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 condition


of mitotic in wild - type fed as ( A ) 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

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

Quantification of 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 condition


of mitotic in wild - type fed as ( A ) 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

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

Quantification of 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


A , B to number of mitotic of dissected gonads from wild - type 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 ) treated with gamma radiation 125 Gy . is expressed as fold changes of positive stained treated over positive untreated Data information : Data are presented as mean ± SD ( A , B N = 2 ( A , B * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . One - way ANOVA Tukey " s multiple comparisons

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

A, B 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 test


Fertilit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated with gamma radiatio at indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified 2 - wa 2 - way ANOVA Tukey " s multiple compa ( C - H )

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

Fertilit 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 of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated wit UV at indicated doses Data information : Data are presented a mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

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 compa


Fertilit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated wit Hydroxyure at indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

Fertilit 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 comparisons


fecundit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated with gamma radiatio at indicated doses Data information : Data are presented a mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

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 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 compa


fecundit of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 , left untreated or treated wit UV at indicated doses Data information : Data are presente as mean ± SE ) , N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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 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


fecundit of wild - type fed 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 indicated doses Data information : Data are presente as mean ± SE N = * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # P < 0 . 05 , # # # P < 0 . 001 , # # P < 0 . 01 , # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified , B ) , 2 - way ANOVA Tukey " s multiple

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

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, 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


A of BRD - 1 representative western blot from wild - type worms fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp - 1 Bar graphs are mean ± Data information : Data are presented as mean ± SD N = 8 - 1 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is

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

A 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 specified


B of mitotic nuclei in single gonad arm of wild - type ( WT ) , brc - 1 ( tm1145 ) brd - 1 ( gk297 ) strains fed as in ( A ) Data information : Data are presented as mean ± SD N = ) , 8 - 1 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is

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

B 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 specified


of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) brd - 1 ( gk297 ) strains fed as in ( A ) left untreated ( C Bar graphs are mean ± . Data information Data are presented a mean ± N = 8 - 10 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification of 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 comparison


of apoptotic corpse in wild - type ( WT ) , brc - 1 ( tm1145 ) brd - 1 ( gk297 ) strains fed as in ( A ) an treated with ionizing radiation 125 Gy ( IR ) ( D ) . Bar graphs are mean ± . Data information : Data are presented a mean ± N = 8 - 10 worms per replicate condition , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 vs untreated ; # # # P < 0 . 001 # # # # P < 0 . 0001 vs con . Not significant if nothing is specified . 2 - way ANOVA Tukey " s multiple

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

Quantification of 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


Kaplan - Meier survival curves of wild - type ( WT ) , ced - 9 ( n1653ts strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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Kaplan-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-1


Kaplan - Meier survival curves of wild - type ( WT ) an ) , brc - 1 ( tm1145 ) ( B strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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Kaplan-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-1


Kaplan - Meier survival curves of wild - type ( WT ) an brd - 1 ( gk297 ) ( C strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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Kaplan-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-1


Kaplan - Meier survival curves of wild - type ( WT ) an glp - 1 ( e2141 ) ( D strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

Kaplan-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-1


Kaplan - Meier survival curves of wild - type ( WT ) an glp4 ( bn2 ) ( E ) , strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against frh - 1 or isp -

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Kaplan-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-1


Kaplan - Meier survival curves of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp - 1 ( F left untreated or treated with 600 , 1200 or 2400 J / m2

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Kaplan-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 UVB


Kaplan - Meier survival curves of wild - type fed transformed with either empty - vector ( con ) or with vector expressing dsRNA agains isp - 1 ( G ) left untreated or treated with 600 , 1200 or 2400 J / m2

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Kaplan-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


A Kaplan - Meier survival curves of wild - type ( WT ) , rrf - 1 ( pk1417 ) , ppw - 1 ( pk2505 ) strains fed transformed with either empty - vector ( con ) or with vector expressing dsRNA against isp -

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

A 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-1


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

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

B 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


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

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


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

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


ChIP - seq tracks of T ( in red ; input is in blue ) around Oct4 ( 7 . 5 kb ) . DE , distal enhancer ; PE , proximal enhancer . T - binding peak around Oct4 - DE is indicated ( arrowhead ) . Right : ChIP - qPCR for enrichment of T on DE regions indicated in Fig EV3E . NC , negative control ( locus without T binding ) . enrichment is indicated by input with SDs ( three independent experiments with two technical replicates ) .

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

ChIP-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 with T - binding elements ( TBEs ) around indicated loci as shown in ( A ) Fig EV3E . Relative luciferase activities of sorted fractions are shown with SDs . ( three independent experiments with two technical replicates ) .

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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).


Left : A representative confocal image of a mouse at E7 . 0 bearing T - GFP Blimp1 - RFP transgenic reporters . Scale bar : 50um . Arrowheads indicate Blimp1 - positive nascent germ in posterior proximal epiblast . Right : FACS of dissociated mouse without visceral endoderm .

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Left: 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 indicated genes measured by qPCR in FACS - sorted ( E ) derived from at different developmental stages ( E7 . 0 - 7 . 5 ) . Ten FACS - sorted in different fractions ( E ) were used for each qPCR with two technical replicates . ∆ Ct values from average Ct values of Arbp Ppia are shown ( n ≥ 3 , log2 scale ) . P values were calculated by two - unpaired t - .

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

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.


ChIP - seq tracks of T ( in red ; input is in blue ) pluripotency factors ( in green ) around Blimp1 ( 100 kb ) Prdm14 ( 100 kb ) . co - bound locus ( L # 1 - 4 ) locus exclusively bound by pluripotency factors but not T ( L # 5 ) are indicated by arrow . NC , negative control ( locus without T binding ) .

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

ChIP-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 enrichment of indicated factors on loci ( L # 1 - 5 ) indicated in ( A ) . enrichment is indicated by input with SDs ( three independent experiments with two technical replicates ) .

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

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 with specific of locus bound by T around L # 1 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 ) .

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

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 Prdm14 measured by qPCR in PGCLCs derived from indicated ESC lines . ∆ Ct values from average Ct values of Arbp Ppia are shown ( log2 scale , mean value of two independent experiments with two technical replicates ) .

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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 ) , Blimp1 - RFP ( red ) Oct4 - GFP ( BROG ) reporter expression ( right ) in gonads of E13 . 5 generated by 4N complementation with indicated ESC lines . Scale bar : 100um . Bottom : of Oct4 - GFP reporter - positive PGCs ( left ) BROG reporter - positive PGCs ( right ) measured by FACS . Blue circles , red squares , green triangles represent of gonadal PGCs in first , second , third independent mutant , respectively , of examined locus ( L # 1 - 5 ) .

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

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).


High constitutive expression of CXCR4 in of SW1417 CRC as measured by flow cytometry .

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High 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 , as measured by ELISA , whereas human control 1BR3 . G fibroblasts express high SDF - 1α , after 48h or 72h of growth in culture . ( N = 2 experiment in duplicate )

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

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 after 1 hour exposure at 1µM , as measured by emission using flow cytometry . ( N = 3 experiments in duplicate ) Significant difference at p = 0 . 002 between T22 - GFP - H6 - FdU AMD3100 + T22 - GFP - H6 - FdU

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

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 groups


Intracellular trafficking of T22 - GFP - H6 - FdU in CXCR4 + SW1417 by confocal microscopy after exposure at 1µM for 24 h . green corresponds to GFP containing nanoconjugates red corresponds to plasma membranes stained with a red dye ( CellMask ™ ) , whereas nucleus was stained in blue with hoescht . insets show detail of intracellular localization of nanostructured , entities , in an isosurface representation within a three - dimensional volumetric x - y - z data

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Intracellular 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 field


Linearized T22 - GFP - H6 - FdU dose - response trend representation compared with unconjugated free oligo - FdU exposure . Antitumor effect was measured as CXCR4 + SW1417 viability by MTT after 72 h exposure as described concentrations . ( N = 3 experiments in duplicate )

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Linearized 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 viability determined by optical microscope of SW1417 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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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).


Selective T22 - GFP - H6 - FdU nanoconjugate biodistribution in subcutaneous CXCR4 + SW1417 CRC tissue 5h after a 100µg single intravenous dose , as measured by emission using IVIS Spectrum 200 ( N = 5 / group ) . Biodistribution is similar to that achieved by T22 - GFP - H6 targeting vector undetectable after Buffer or free oligo - FdU treatment . ( N = 5 mice / group )

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

Selective 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 T22 - GFP - H6 - FdU ( green ) CXCR4 receptor ( red ) release of T22 - GFP - H6 - FdU into cytosol in CXCR4 + 5h after a 100ug dose of nanoconjugate , as measured by dual anti - GFP / anti - CXCR4 immunofluorescence ( IF ) . DAPI ( blue ) . emission was measured in green red channels using Image J software expressed as mean ( A ) ± s . e . m ( µm2 ) ( N = 10 , 2 fields x 5 mice ; 200X ) . Note significant ( P = 0 . 003 ) increase in occupied by green dots ( nanoconjugate realeased to cytosol ) in T22 - GFP - H6 - FdU - treated tumors , compared to free oligo - FdU - treated control tissues . Scale bar , 50 µm .

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

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 CXCR4 antagonist AMD3100 completely blocks T22 - GFP - H6 - FdU biodistribution , as measured by emission . is not detected in Buffer or free oligo - FdU controls ( N = 5 fields / group ) .

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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).